Evaluation 3

Cloning Expression Purification and Enzymological Characterization of NS2BNS3 Protease RNA Helicase

protein of Japanese Encephalitis Virus

Chakard Chalayut Advisor Asst Prof Gerd Katzenmeier PhD

Laboratory of Molecular Virology Institute of Molecular Biology amp Genetics

Japanese Encephalitis Virus (JEV)

-Flaviviridae family-Mosquito-borne neurotropic flavivirus

bullcauses severe central nerve system diseases


Culex tritaeniorhynchus

Source fehdgovhk



Source vietnammedicalpracticecom



JEV causes severe central nerve system d i s e a s e s s u c h a s po l iomye l i t i s - l i ke acute flaccid paralysis aseptic meningitis and encephalitis

Sourcewondercdcgov




50000 Cases


30 fatality rate


10000 Cases

Prevention and treatment of JEV disease


Drug No drug exist


Drug No drug exist

Vaccine development Available vaccine


Drug No drug exist

Vaccine development

Mosquitoes control Elimination of mosquitoes breeding places

Available vaccine

Molecular biology of Japanese Encephalitis Virus

The NS2Bbull 130 aabull activating domain central hydrophilic region (Falgout et al 1993)bull 3 membrane spanning parts

The NS2B

hydrophobicity plot

bull 130 aabull activating domain central hydrophilic region (Falgout et al 1993)bull 3 membrane spanning parts

The NS2B

hydrophobicity plot

51 DMWLERAADISWEMDAAITGSSRRLDVKLDDDGDFHLIDDPGVP 95


The NS2BHypothetical model NS2B-NS3 complex

hydrophobicity plot


Brinkworth et al 1999


The NS3

Theoretical model from PDB 2I84

The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3

bullChymotrypsin-like fold2-β barrel domains bullInactive alonebullEnzymersquos pocket is small


The NS3 protease

The NS3 protease

bullNS3 serine protease domain 20 kDabullcatalytic residues His51 Asp75 Ser135

Background

bull Lin C W et al2007

Backgroundbull Ser46 to Ile60 were essential region required for NS3

protease activity

bull Ala substition of Trp50 Glu55 and Arg56 in NS2B shown significantly reduced NS3 protease activity


Objective

Objective

bull to perform cloning of the NS2B-NS3 portion of the JEV polyprotein express in Ecoli and biochemically purify to determinants of clevage activity and cofactor requirement will be analyzed and compared to dengue virus

bull The second objective is to study differences in substrate specificity and inhibitors by using peptide subst ra tes incorporated wi th fluorogenic or chromogenic reported groups

Method amp Result pLS with NS2B-NS3 JEV


NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease

Figure 2 The PCR product NS3protease JEV amplified from NS2B-NS3 JEV (Lane 3 to 5 ) The size of NS3 protease was 594 bp

Figure 1 The PCR product NS2B(H) JEV amplified from NS2B-NS3 JEV (Lane 3 and 4) The size of NS2B(H) was 187 bp

1500

1000900800

700

600

500

400

300

Figure 3 The SOE-PCR product NS2B(H)-NS3protease JEV (Lane 2 to 5 ) The size of NS2B(H)-NS3 protease was 765 bp

Control

1500

1000900800700

600

500

400

300

200

100

Figure 4 The NS2B(H)-NS3protease JEV (Lane 3 ) after digested with BamHI and KpnI The size of NS2B(H)-NS3 protease was 765 bp

Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result

NS2B(H)-NS3p JEV pTrcHis A

Ligation amp Transformation

Site Screening amp Digest with Restriction Enzyme

Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2

Digest with Restriction Enzyme

3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed

bullLane 1 λBstII marker

bullLane 2 Clone 1 with BamHI and KpnI digerstion

bullLane 3 Clone 1 with BamHI digestion

bullLane 4 Clone 1 without digestion

Sequencing of candidate clone

Deduced amino acid sequences of Japanese encephalitis virus

NS2B(H) C-terminalNS2B(H)

NS3p

Expression of Japanese encephalitis virus NS2B(H)NS3 protease at different time

0 Hr

2 Hr

4 Hr

6 Hr

8 HrOD= 04-06 induction by IPTG

incubate overnight

1

SDS-PAGE Analysis of Japanese encephalitis virus NS2B(H)NS3 protease at different time

210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

ptrc 0- 8 Hr NS2B(H)NS3p 0- 8 Hr

NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD

29 kDNS2B(H)NS3 protease

NS3

NS2B(H)

Western blot analysis of Japanese encephalitis virus NS2B(H)NS3 protease at different time

Expression of Japanese encephalitis virus NS2B(H)NS3 protease expressed at different temperatures

OD= 04-06 induction by IPTGIncubate 8 Hrs

incubate overnight

1 18 ˚C

37 ˚C

25 ˚C

SDS-PAGE analysis of Japanese encephalitis virus NS2B(H)NS3 protease expressed at different temperatures

pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)

Western blot analysis of Japanese encephalitis virus NS2B(H)NS3 protease expressed at different temperatures

mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)

Expression of Japanese encephalitis virus NS2B(H)NS3 protease expressed at different IPTG concentrations


incubate overnight

1

01 mM02 mM03 mM04 mM

08 mM07 mM06 mM

05 mM

SDS-PAGE analysis of Japanese encephalitis virus NS2B(H)NS3 protease expressed at different IPTG concentrations

210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)

Western blot analysis of Japaneseencephalitis virus NS2B(H)NS3 protease expressed at different IPTG concentrations

mar

ker

358 kD


01m

M

08m

M

Expression of Japanese encephalitis virus NS2B(H)NS3 protease at small scale expression

OD= 04-06 induction by o1 mM IPTG

Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis

Sonication and Collect

fraction


SDS-PAGE analysis of Japanese encephalitis virus NS2B(H)NS3 protease at small scale expression

210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker

Lane 1 maker Lane 2 Control pTrcHisLane 3 Total fraction (T)Lane 4 Insoluble fraction (I)Lane 5 Soluble fraction (S)

NS2B(H)NS3 protease

NS3 protease

NS2B(H)

Western blot analysis of Japanese encephalitis virus NS2B(H)NS3 protease at small scale expression

IT S

NS2B(H)NS3 protease358 kD


210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker

SDS-PAGE analysis of Japanese encephalitis virus NS2B(H)NS3 protease at large scale expression

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)

Lane 1 maker Lane 2 Total fraction (T)Lane 3 Insoluble fraction (I)Lane 4 Soluble fraction (S)

Western blot analysis of Japanese encephalitis virus NS2B(H)NS3 protease at large scale expression

mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)


Purification of the NS2B(H)NS3p protein harboring the N-terminal polyhistidine tag

The NS2B(H)NS3 protease was purify by HiTrap Chelating column

Method

Soluble fraction in buffer H

Wash with Buffer H (30 mM imidazole)

Elute with Buffer H (100 mM imidazole)

SDS-PAGE analysis of Japanese encephalitis virus NS2B(H)NS3 protease from Hi-trap purification

21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion

Western-blot analysis of Japanese encephalitis virus NS2B(H)NS3 protease from Hi-trap column purification

NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion

Characterize the activity of NS2B(H)NS3p JEV with Ac-RRRR-pNA

The substrate Ac-RRRR-pNA is based on the para-nitroanilide principle

the enzyme will cleave between the tetra arginine and release pNA

Free pNA will be monitored at A405 by the spectrophotometry method The change of pNA will change the color of buffer and correlate to the activity of the enzyme

Result

Trypsin

NS2B(H)NS3p JEV

substrate

NS2B(H)NS3p JEV = 10 μM trypsin = 04 μM substrate = 500 μM

Problem

bull The protein has by products from the purification step

bull No activity

bull The fusion protein was different from the previous work of JEV

The International Journal of Biochemistry amp Cell Biology 39 (2007) 606ndash614

Method

Soluble fraction in buffer A

Wash with Buffer A (30 mM imidazole)

Elute with Buffer A (100 mM imidazole)

(50mM Hepes pH 70 500mM NaCl)

Method

The eluted protein

(superdex 75 HR 10300)

peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate

Protein assay method7-Amino-4-Methyl Coumarin (AMC) standard curve

bull A serial dilution of AMC from 3 μM - 50 μM was prepared in 100 μl assay buffer (200 mM Tris-HCl pH 95 135 mM NaCl and 30 Glycerol)

bull incubated at 37 C for 30 minutes

bull Fluorescence signals were measured at an excitation wavelength 355 nm and emission 460 at 37 C nm Fluorescence signals was plotted against AMC concentration

result

Data 1

0 20 40 600

20000

40000

60000

80000

100000Fluorescence unit

[AMC] (microM)

Flu

ore

ce

nce

un

its

The correlation between units with AMC concentrations was calculated from 1

slope of linear regression 1 fluorescence unit = 6839 nM [AMC]

Protein assay methodEnzyme Activity assay

NS2B(H)-NS3p JEV protein 100 μl assay buffer (200 mM Tris-HCl pH 95

135 mM NaCl and 30 Glycerol)


adding 200 μM of Pyr-RTKR-AMC substrate

bull Fluorescence signals were measured at an excitation wavelength 355 nm and emission 460 at 37 C nm and monitored every 3 minutes for 2 hr

resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it

Progress curves for enzymes-catalyzed hydrolysis observed at 200 μM of Pyr-RTKR-AMC were plotted

between fluorescence signals against time

Protein assay methodDetermination of kinetic parameters

Pyr-RTKR-AMC was varied the concentrations from 15 μM to 500 μM




bull Fluorescence signals were measured at an excitation wavelength 355 nm and emission 460 at 37 C nm and monitored for 2 hr

resultData 1

0 200 400 6000

500

1000

1500

2000

[Pyr-RTKR-AMC] (microM)

V

elo

city

(n

Mm

in)

Vmax (nMmin) Km (microM) Kcat (s-1) Kcat Km(M-1s-1)

2672 2264 322 0014

Whatrsquos next

bull Try to improve purification and find the amount of active protein

bull compare to Den NS2B(H)-NS3p


-Flaviviridae family-Mosquito-borne neurotropic flavivirus

bullcauses severe central nerve system diseases



Source fehdgovhk







Sourcewondercdcgov




50000 Cases


30 fatality rate


10000 Cases



Drug No drug exist


Drug No drug exist



Drug No drug exist

Vaccine development


Available vaccine



The NS2B

hydrophobicity plot


The NS2B

hydrophobicity plot




hydrophobicity plot




The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next





Source fehdgovhk







Sourcewondercdcgov




50000 Cases


30 fatality rate


10000 Cases



Drug No drug exist


Drug No drug exist



Drug No drug exist

Vaccine development


Available vaccine



The NS2B

hydrophobicity plot


The NS2B

hydrophobicity plot




hydrophobicity plot




The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next









Sourcewondercdcgov




50000 Cases


30 fatality rate


10000 Cases



Drug No drug exist


Drug No drug exist



Drug No drug exist

Vaccine development


Available vaccine



The NS2B

hydrophobicity plot


The NS2B

hydrophobicity plot




hydrophobicity plot




The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next








Sourcewondercdcgov




50000 Cases


30 fatality rate


10000 Cases



Drug No drug exist


Drug No drug exist



Drug No drug exist

Vaccine development


Available vaccine



The NS2B

hydrophobicity plot


The NS2B

hydrophobicity plot




hydrophobicity plot




The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next






Sourcewondercdcgov




50000 Cases


30 fatality rate


10000 Cases



Drug No drug exist


Drug No drug exist



Drug No drug exist

Vaccine development


Available vaccine



The NS2B

hydrophobicity plot


The NS2B

hydrophobicity plot




hydrophobicity plot




The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next





Sourcewondercdcgov




50000 Cases


30 fatality rate


10000 Cases



Drug No drug exist


Drug No drug exist



Drug No drug exist

Vaccine development


Available vaccine



The NS2B

hydrophobicity plot


The NS2B

hydrophobicity plot




hydrophobicity plot




The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next






50000 Cases


30 fatality rate


10000 Cases



Drug No drug exist


Drug No drug exist



Drug No drug exist

Vaccine development


Available vaccine



The NS2B

hydrophobicity plot


The NS2B

hydrophobicity plot




hydrophobicity plot




The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next





50000 Cases


30 fatality rate


10000 Cases



Drug No drug exist


Drug No drug exist



Drug No drug exist

Vaccine development


Available vaccine



The NS2B

hydrophobicity plot


The NS2B

hydrophobicity plot




hydrophobicity plot




The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




50000 Cases


30 fatality rate


10000 Cases



Drug No drug exist


Drug No drug exist



Drug No drug exist

Vaccine development


Available vaccine



The NS2B

hydrophobicity plot


The NS2B

hydrophobicity plot




hydrophobicity plot




The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




30 fatality rate


10000 Cases



Drug No drug exist


Drug No drug exist



Drug No drug exist

Vaccine development


Available vaccine



The NS2B

hydrophobicity plot


The NS2B

hydrophobicity plot




hydrophobicity plot




The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




10000 Cases



Drug No drug exist


Drug No drug exist



Drug No drug exist

Vaccine development


Available vaccine



The NS2B

hydrophobicity plot


The NS2B

hydrophobicity plot




hydrophobicity plot




The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next





Drug No drug exist


Drug No drug exist



Drug No drug exist

Vaccine development


Available vaccine



The NS2B

hydrophobicity plot


The NS2B

hydrophobicity plot




hydrophobicity plot




The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




Drug No drug exist


Drug No drug exist



Drug No drug exist

Vaccine development


Available vaccine



The NS2B

hydrophobicity plot


The NS2B

hydrophobicity plot




hydrophobicity plot




The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




Drug No drug exist



Drug No drug exist

Vaccine development


Available vaccine



The NS2B

hydrophobicity plot


The NS2B

hydrophobicity plot




hydrophobicity plot




The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




Drug No drug exist

Vaccine development


Available vaccine



The NS2B

hydrophobicity plot


The NS2B

hydrophobicity plot




hydrophobicity plot




The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next





The NS2B

hydrophobicity plot


The NS2B

hydrophobicity plot




hydrophobicity plot




The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




The NS2B

hydrophobicity plot


The NS2B

hydrophobicity plot




hydrophobicity plot




The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



The NS2B

hydrophobicity plot


The NS2B

hydrophobicity plot




hydrophobicity plot




The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



The NS2B

hydrophobicity plot




hydrophobicity plot




The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




hydrophobicity plot




The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



The NS3


The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



The NS3

Protease


The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



The NS3

NTPase

Protease

RNA Helicase


The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



The NS3



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



The NS3 protease

The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



The NS3 protease


Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



Background



protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




protease activity



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



Objective

Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



Objective





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next





NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




NS2B(H) NS3p


NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




NS2B(H) NS3p

SOE-PCR


NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



1500

1000900800700

600

500

400

300

200

Control

NS

2B(H

)

NS

2B(H

)

1500

1000900800

700

600

500

400

300

200

Control

NS

3 protease

NS

3 protease

NS

3 protease



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



1500

1000900800

700

600

500

400

300


Control

1500

1000900800700

600

500

400

300

200

100


Control

NS

2B(H

)-NS

3p

NS2B(H)-NS3p

Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



Method amp Result

Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



Method amp Result




Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



Size Screening

2313 kb

942 kb 656 kb 456 kb

232 kb 203 kb

pTrc

pla

smid

Con

trol

Clo

ne 1

Clo

ne 3

Clo

ne 4

Clo

ne 5

Clo

ne 6

Clo

ne 7

Clo

ne 8

Clo

ne 9

Clo

ne 1

0C

lone

11

Clo

ne 1

2C

lone

13

Clo

ne 1

4

Clo

ne 1

5C

lone

16

Clo

ne 1

7

Clo

ne 2


3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




3645 kb

2323 kb 1929 kb

1371 kb 1264 kb

702 bp

4324 kb4822 kb5686 kb6369 kb7242 kb8454 kb

clon

e 1

with

Bam

HIK

pnI

clon

e 1

with

Bam

HI

clon

e 1

undi

gest

ed








NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next






NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next





NS3p


0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




0 Hr

2 Hr

4 Hr

6 Hr


incubate overnight

1


210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




210 kD125 kD101 kD562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker


NS2B(H)NS3

NS3

NS2B(H)

mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



mar

ker

NS2B(H)NS3p 0- 8 Hr

358 kD

21 kD


NS3

NS2B(H)




incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next





incubate overnight

1 18 ˚C

37 ˚C

25 ˚C


pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




pTrc

37

˚C

pTrc

30

˚C

pTrc

0 h

r 37

˚C

pTrc

25

˚C

mar

ker

JEV

37

˚C

JEV

25

˚C

JEV

18

˚C

JEV

30

˚C

pTrc

18

˚C

210 kD125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




mar

ker

pTrc

0 h

r 37

˚C

pTrc

25

˚C

pTrc

18

˚C

NS

2B(H

)NS

3p 1

8 ˚C

NS

2B(H

)NS

3p 3

7 ˚C

NS

2B(H

)NS

3p 2

5 ˚C

NS

2B(H

)NS

3p 3

0 ˚C

pTrc

37

˚C

pTrc

30

˚C

101 kD

358 kD

69 kD

21 kD

NS2B(H)NS3

NS3 protease

NS2B(H)



incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next





incubate overnight

1


08 mM07 mM06 mM

05 mM


210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




210 kD125 kD

101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

01m

M

08m

M

NS2B(H)NS3

NS3 protease

NS2B(H)


mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




mar

ker

358 kD


01m

M

08m

M



Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next





Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




Incubate 8 Hrs

incubate overnight

1 Centrifuge Lysis


fraction



210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




210 kD125 kD

ITC S

101 kD

562 kD

358 kD

29 kD

69 kD

21 kD

mar

ker


NS2B(H)NS3 protease

NS3 protease

NS2B(H)


IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




IT S



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



210 kD

125 kD101 kD

562 kD

358 kD

29 kD

21 kD

69 kD

mar

ker

mar

ker


T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)



mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




mar

ker

mar

ker

T SI T SI

NS2B(H)NS3 protease

NS3 protease

NS2B(H)




Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next





Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



Method





21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




21 kD

NS2B(H)NS3 protease

NS3 protease

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion


NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




NS2B(H)

NS2B(H)NS3

NS3 protease

21 kD

69 kD

358 kD

29 kD

mar

ker

Flow

trou

gh

indu

ctio

n

undu

ctio

n

Was

hing

frac

tion

Elu

te a

nd c

once

ntra

te

fract

ion

Sol

uble

frac

tion





Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next







Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



Result

Trypsin

NS2B(H)NS3p JEV

substrate


Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



Problem


bull No activity



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next




Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



Method





Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



Method

The eluted protein


peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



peak

2

peak

1

result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



result

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

peak

2

peak

1

Solu

ble

Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



Flow

thro

ugh

wash

Elut

e

Solu

ble

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

unin

duct

ion

Gel

filtr

atio

n

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



21 kD

69 kD

210 kD125 kD

562 kD

101 kD

358 kD

29 kD

Flow

thro

ugh

wash

Elut

e

Solu

ble

indu

ctio

n

Gel

filtr

atio

n

The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



The substrate

The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



The substrate





result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next







result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



result

Data 1

0 20 40 600

20000

40000

60000

80000


[AMC] (microM)

Flu

ore

ce

nce

un

its









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next









resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



resultData 1

0 50 100 150

0

10000

20000

30000Control

200 mM

time

Flu

ore

sce

nce

un

it









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next









resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



resultData 1

0 200 400 6000

500

1000

1500

2000


V

elo

city

(n

Mm

in)


2672 2264 322 0014

Whatrsquos next



Whatrsquos next



Evaluation 3

Education

ns3 jev lane

ns2b jev lane

ns2bns3 figure

size of ns3 protease

undigested clone

jev polyprotein

ns2bns3 portion

ns3 protease theoretical