R Pharmaceuticals J. Stracke, 09/01 Rapid production of fusion proteins using the RTS 100/500 Systems Jan Stracke, Michael Schräml, Andreas Junger, Dorothee.

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Rapid production of

fusion proteins using

the RTS 100/500

SystemsJan Stracke, Michael Schräml, Andreas Junger, Dorothee Ambrosius

and

Martin Lanzendörfer

Roche Diagnostics GmbH, Pharmaceutical Research, Dept. of Biochemistry, Penzberg,

Germany

J. Stracke, 09/01

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ticalsProtein Production

High need for purified proteins in

pharmaceutical research for:

X-ray crystallography (X-ray)

Nuclear Magnetic Resonance (NMR)

High Throughput Screening (HTS)

Therapeutic Proteins (e.g. antibodies)

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ticalsBottlenecks of protein crystallisation

Gene ProteinCrystal/

Structure

Screening of various

constructs for expression

Screening of various muteins and/or truncated

constructs for crystallisation

J. Stracke, 09/01

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ticalsClassical Protein Production

cloning

expression

fermentation

cell lysis

soluble / insoluble protein

refolding

purification

characterisation

activity assays, interaction analysis

high need for acceleration of protein production

high need for acceleration of methods

time c

on

su

min

g s

tep

J. Stracke, 09/01

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ticalsRapid Translation System (RTS)

gene

DNA-templateT7-RNA polymerase E.coli lysate

proteinmRNA

gene

RTS 100 RTS 500 RTS 500 HY

50 µg / ml 500 µg / ml 5 mg / ml

control of expression by SDS-PAGE, Western blotting, ELISA

transcription & translation

RTS 500

RTS 100

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ticalsProteins successfully expressed with RTS

Endostatin

Erythropoietin

Interleukin-2

p40phox SH3 mod.

Phosphodiesterase

Rab 5

VEGF-receptorGreen fluorescent p.b -Galactosidase

HIV Tat

s-Adenosylmethio-nine synthetase 1

Protein MW20

30

15.4

47

56

22

41

28

118

10

41.6

Organism

human

human

human

human

human

human

human

animal

bacteria

virus

yeast

Function

Inhibitor

hormone

hormone

regulatory protein

regulatory protein

GTPase

receptor fragment

fluorescent protein

enzyme

transcription factor

enzyme

Yield RTS 500 [µg]

100

550

600

90

100

100

250

560

105

n. d.

6

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ticalsSuccessfully expressed proteins - a system comparison

RTS 500

vs

RTS 500 HY

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ticalsFurther applications of RTS

Expression of...

...cell toxic proteins… ...two proteins in parallel...

…in the RTS 500.

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The protein was expressed using the RTS 500 E. coli HY Kit. Approximately 3 mg of PSP protein, fused at the C-terminus with a His6 tag, was produced in a single 1 ml reaction,

purified, concentrated and subsequently crystallised at 25°C using the hanging-drop method. Data courtesy of Ho S. Cho, Weiru Wang, Sung-Hou Kim and David E. Wemmer, Berkeley Structural Genomic Center.

Crystals of phosphoserine phosphatase (PSP)

Successfully crystallised proteins

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ticals RTS 100 expression

• in vitro batch expression in 50 µl reactions

• designed for the use of linear PCR-generated DNA templates

• enables the fast and parallel expression and evaluation of numerous constructs (e.g. for crystal engeneering)

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gene X

T7P RBS tag cs

PCR

cs tag T7T

gene X cs tag T7TT7P RBS tag cs

promotor module terminator module

linear template for in vitro translation (batch)

Various promotor and terminator modules can be prepared and fused by PCR in numerous combinations with the gene of interest. Expression of these constructs is performed in the cell-free translation system RTS 100. MTP format is possible!

Schematic illustration of template generation for RTS 100 expression using PCR

RTS 100 expression

J. Stracke, 09/01

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ticalsSuccessfully expressed proteins - a system comparison

RTS 100 HY vs RTS 500 HY

lower yields in the RTS 100 compared to RTS 500

J. Stracke, 09/01

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ticalsHigh Throughput Protein Production (HTPP)

using RTS 100

goal acceleration of protein production

PCR product (e.g. RT-PCR)

RTS (in vitro expression)

soluble / insoluble protein

refolding

high affinity purification

activity assays, interaction analysis

production in days (not

months)

high throughput

high flexibility

automation

small scale production

need for high yield

expression

classical or in vitro

optimisation

MTP

-96 w

ell-fo

rmat

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Evaluation of:

•Solubility

•Affinity-tags (purification, immobilisation)

•Ligand activity (BIAcore)

using the RTS 500 system for construct expression.

Example 1

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ticalsExample 1 - Structures of MMP2-PEX and TIMP2

MMP2-PEX: hemopexin-like domain of MMP2MW = 23 kDa; 1 disulfide bridge

TIMP2: tissue type inhibitor of matrixmetallo-proteinase 2MW = 22 kDa; 6 disulfide bridges

Gohlke et al. (1996)

PEX (MMP2)

1

23

4

1

23

4

N

C

TIMP2

Williamson et al. (1994)

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4 protein coding genes in 7 constructs

promotor fused genes

tac PinPoint-tag PEX2

PinPoint-tag TIMP2tac

PinPoint-tag Protein Atac

AVITAG PEX2T7

Protein B AVITAGT7

Strep-tag PEX2T7

Poly-Glu--tag PEX2T7

E.coli

RTS-500andE.coli

vector

Xa3-PinPoint

Xa3-PinPoint

Xa3-PinPoint

pIVEX 2.1

pIVEX 2.1

pIVEX 2.2b Nde

pIVEX 2.3 MCS

Example 1 - Evaluation of constructs for BIAcore analysis

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Relative yields of expressed protein - Improvement of solubility in the RTS-500 System

E.coli RTS-500 System

0 %

Protein A NtPP

Protein B

NtAT

Example 1 - Evaluation of constructs for BIAcore analysis

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-10

0

10

20

30

40

50

60

70

80

-50 0 50 100 150 200 250 300 350 400

Time [s]

Resp

on

se Differen

ce [RU

]

Association Dissociation

Ligand : PEX2_NtAT Analyte : TIMP2Injection

Equilibrium constant KD = 1.5x 10-10M

TIM

P-2

con

cen

tratio

n

Analysis of ligand interactions of RTS-expressed PEX2-NtAT

(N-terminaler AVITAG) with TIMP-2 using BIAcore

Example 1 - Evaluation of constructs for BIAcore analysis

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ticalsExample 2 - Production of active Protein B

Evidence exists that:

• a truncated form may be active or

• co-factors may be required for activity

These aspects are being addressed using the RTS 100

and 500 systems for construct expression/evaluation.

Aim: Production of sufficient quantities of active Protein B for biochemical characterisation and crystallisation.

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ticalsExample 2 - Production of active Protein B

truncated/mutated constructs by PCR

activity assay

expression in 96 well plates (RTS 100)

target protein expression(RTS 500)

immobilisation

ligand fishing

co-expression withselected

activating proteins(e.g. RTS 100)

activity assay

RT-PCR

ligand identification

ActiveProtein B

Truncations/mutations Co-expression

J. Stracke, 09/01

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ticalsExample 2 - Production of active Protein B

Truncated Protein B constructs, generated by PCR

for expression in the RTS 100

Prot. B

gene B cs tag T7TT7P RBS tag cs

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ticalsExample 3 - Production of various muteins

Aim: Evaluation of approx. 50 muteins of Protein C (extracellular, 7 disulphide bonds) for crystallisation.

Strategy: generation of numerous mutated linear constructs by PCR

assay for ligand activity (e.g. BIAcore)

selection of active and inactive constructs forlarge scale expression and

crystallisation

expression in 96 well plates (RTS 100)

refolding and purification in 96 well plates

info about ligand binding site

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• proteins of interest can be expressed in an active, soluble form in the RTS systems in µg/mg quantities for initial characterisation (e.g. activity)

• proteins can easily be co-expressed if necessary

• due to the possibility to use linear PCR products as templates, the RTS 100 allows rapid parallel screening of numerous protein constructs with regard to activity, ligand binding and solubility

• RTS 100 is predestinated for automation of protein expression and purification

Conclusions

Overcoming the bottleneck of protein production for crystallography seems possible in the near future using RTS!

J. Stracke, 09/01

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ticalsAcknowledgements

Roche Pharma Research

A. Grossmann

R. Engh

M. Dangl

P. Rüger

K. Lang

Roche Mol. Biochemicals

C. Nemetz

A. Gräntzdörfer

S. Wessner

M. Watzle

Max Planck Inst.f. Biochemie

R. Engh

R. Huber

N. Heim

M. Wisniewska

TU-München

J. Buchner