Protein Purification Involving a Unique Auto-Cleavage Feature of a Repeated EAAAK Peptide

Protein Purification Involving a Protein Purification Involving a Unique Auto-Cleavage Feature of Unique Auto-Cleavage Feature of a Repeateda Repeated EAAAK Peptide EAAAK Peptide

Enzyme ResearchEnzyme Research

Protein Protein ProductionProduction

Bio-conversionBio-conversion

Bio-medical applicationBio-medical application

Structure & Structure & CatalyticCatalytic

MechanismMechanism

Interdisciplinary Research of Interdisciplinary Research of Enzyme TechnologyEnzyme Technology

Enzyme ResearchEnzyme Research

Protein Protein ProductionProduction

Bio-conversionBio-conversion

Bio-medical applicationBio-medical application

Structure & Structure & CatalyticCatalytic

MechanismMechanism

Interdisciplinary Research of Interdisciplinary Research of Enzyme TechnologyEnzyme Technology

Expression systems

Purification Techniques

Oligosaccharide preparation

Biomass degradation

Transglycosylation

LC/MS/MSBiosensor/SPR

SiNW-FETNano-particles

Glycoside hydrolases GH-1, GH-3, GH-17, GH-18,GH-29, GH-46, GH-54, GH-64, GH-72, GH-75

General Flow Chart of General Flow Chart of PurificationPurificationAnimal tissueAnimal tissue Plant materialsPlant materials MicroorganismsMicroorganisms

ExtracellularExtracellularenzymesenzymes

IntracellularIntracellularenzymesenzymes

DisruptionDisruptionFiltrationFiltration

ConcentrationConcentration PurificationPurification Pure EnzymePure Enzyme

FermentationFermentation

ExtractionExtraction

GrindingGrindingGrindingGrinding

Strategy for massive production and Strategy for massive production and purification of proteinpurification of protein

Current strategies and problemsCurrent strategies and problems

Recombinant protein technology is the best Recombinant protein technology is the best solution so far. solution so far.

Current strategies and problemsCurrent strategies and problems

To simplify purification of recombinant proteins, To simplify purification of recombinant proteins, several engineered affinity tags several engineered affinity tags are used with which fusion protein can be purified to near fusion protein can be purified to near homogeneity in a simple procedure.homogeneity in a simple procedure.

LinkerLinker

Carrier ProteinCarrier Protein Target proteinTarget protein

Protein purification based on affinity bindingProtein purification based on affinity binding

BindingBinding

LinkerLinker

Carrier ProteinCarrier ProteinAffinity matrixAffinity matrix Target proteinTarget protein

Protein purification based on affinity Protein purification based on affinity bindingbinding

• Glutathione S-transferase (Novagen, GST)Glutathione S-transferase (Novagen, GST)• Maltose-binding protein (pMAL system, NEB) Maltose-binding protein (pMAL system, NEB) • Chitin-binding domain (IMPACT system, NEB)Chitin-binding domain (IMPACT system, NEB)

BindingBinding LinkerLinker

Carrier ProteinCarrier ProteinAffinity matrixAffinity matrix Target proteinTarget protein

Protein purification based on affinity bindingProtein purification based on affinity binding

washwashexcessexcess

Dialysis to remove

Current strategiesCurrent strategies

Carrier protein (or Tag) may need to be removed, Carrier protein (or Tag) may need to be removed, commonly by proteasecommonly by protease, after fusion protein has been purified before subsequent use in downstream application.

• Costly affinity matrix required.Costly affinity matrix required.

• Post proteolytic process needed. Post proteolytic process needed.

LinkerLinker

Affinity matrixAffinity matrix

Common DrawbacksCommon Drawbacks

Protein purification based on affinity bindingProtein purification based on affinity binding

• Glutathione S-transferase (Novagen, GST)Glutathione S-transferase (Novagen, GST)• Maltose-binding protein (pMAL system, NEB) Maltose-binding protein (pMAL system, NEB) • Chitin-binding domain (IMPACT system, NEB)Chitin-binding domain (IMPACT system, NEB)• Chitin-binding protein with auto-cleavage peptide linkerChitin-binding protein with auto-cleavage peptide linker (developed by NCTU)(developed by NCTU)

BindingBinding LinkerLinker

Carrier ProteinCarrier ProteinAffinity matrixAffinity matrix Target proteinTarget protein

A vector containing A vector containing chitin-binding proteinchitin-binding protein and and repeated EAAAKrepeated EAAAK peptide linker to form peptide linker to form a simple and cost-effective system for a simple and cost-effective system for

protein expression and purification.protein expression and purification.

A new system developed by our groupA new system developed by our group

MCSMCSLinkerLinkerCBPCBP

Repeated EAAAK peptide Repeated EAAAK peptide with auto-cleavage propertywith auto-cleavage property

Characteristics of chitin-binding Protein (CBP)Characteristics of chitin-binding Protein (CBP) • CBP promotes the hydrolysis of chitin catalyzed CBP promotes the hydrolysis of chitin catalyzed

by chitinase.by chitinase.• CBP has good binding specificity for chitin.CBP has good binding specificity for chitin.• pHpH ＞＞ 8, CBP can bind to chitin.8, CBP can bind to chitin.• pHpH ＜＜ 6, CBP can be eluted.6, CBP can be eluted.

History of our finding……History of our finding……

Starting from the study of Chitinase from Starting from the study of Chitinase from Bacillus NCTU2Bacillus NCTU2

Structures of ChitinasesStructures of Chitinases

TIM-barrel structureTIM-barrel structure

Chitin-binding domain Chitin-binding domain (1~150 aa)(1~150 aa)

LinkerLinker

CBPCBPCatalytic Catalytic

active ?active ?

Bacillus Bacillus NCTU2 NCTU2 ChiAChiA

Serratia ChiASerratia ChiA

protease cutting site

Nde1

PT7CBP21 gene Linker 1~7 protease cutting site

Nde1

PT7CBP21 gene Linker 1~7 protease cutting site

Nde1

PT7CBP21 gene Linker 1~7

A

protease cutting site

Nde1

PT7CBP21 gene Linker 1~7 protease cutting site

Nde1

PT7CBP21 gene Linker 1~7 protease cutting site

Nde1

PT7CBP21 gene Linker 1~7

A

AKRGWIGTGGEGPGGGTGGEGPGGGGPGEGGTGTTLGSNLGTLGLK(EAAAK)5GTGGEGPGGGGPGEGGTGGTGGEGPGG(GGGGS)5

1234567

amino acid sequenceLinker

AKRGWIGTGGEGPGGGTGGEGPGGGGPGEGGTGTTLGSNLGTLGLK(EAAAK)5GTGGEGPGGGGPGEGGTGGTGGEGPGG(GGGGS)5

1234567

amino acid sequenceLinker

Vector designVector design

Procedure of pRSET/CBP-V5G vector constructionProcedure of pRSET/CBP-V5G vector construction

MCSCBP Linker Protease cutting site

LinkerLinker

CBPCBP

The chimeric chitinase is active The chimeric chitinase is active without significant improvement in without significant improvement in catalysis.catalysis.

However, interestingly……However, interestingly……

The fusion protein broke into The fusion protein broke into two fragments at pH 6.0!two fragments at pH 6.0!

Lane 1Lane 1 ：： Sample kept in water for hours (pH Sample kept in water for hours (pH 6.9)6.9)..

Lane 2Lane 2 ：： Sample in Pi buffer (50 mM, pHSample in Pi buffer (50 mM, pH 6.0)6.0) for for 11 day day

Lane 3Lane 3 ：： Sample in Pi buffer (50, mM, pHSample in Pi buffer (50, mM, pH 6.0) 6.0) for for 22 days days

Lane 4Lane 4 ：： Sample kept in water (pH Sample kept in water (pH 6.9) for 6.9) for 11 day day

Lane 5Lane 5 ：： Sample kept in water (pH Sample kept in water (pH 6.9) for 6.9) for 22 day day

CBP-V5G-ChiA (MW 58 kDa)CBP-V5G-ChiA (MW 58 kDa)

NCTU2 ChiA (36 NCTU2 ChiA (36 kDa)kDa)

CBP (19 kDa) CBP (19 kDa)

The fusion protein broke into two fragments at pH The fusion protein broke into two fragments at pH 6.0!6.0!

22.5

CBP-V5G-CNS( 45 kDa)

Exchange buffers with pH 4.2 - Exchange buffers with pH 4.2 - 8.0 and kept at 25 for 12 h.℃8.0 and kept at 25 for 12 h.℃

CBP (19 kDa)

CNS (chitosanase, 24kDa)

Other cases

Dose Auto-cleavage occur on Dose Auto-cleavage occur on CBP-(EAAAK)CBP-(EAAAK)55-G-ChiA? -G-ChiA?

Or contamination of protease?Or contamination of protease?

pH-dependent auto-cleavage of (EAAAK)pH-dependent auto-cleavage of (EAAAK)55 linker!! linker!!

M pH8.0 7.5 7.0 6.0 5.0 4.2

CBP-V5G-CNS ( 45kDa)CBP-V5G-CNS ( 45kDa)

100 for 10 min under pH ℃100 for 10 min under pH ℃3.63.6 ；； exchange buffers with exchange buffers with pH 4.2 - 8.0 and then kept at pH 4.2 - 8.0 and then kept at 25 for 12 h.℃25 for 12 h.℃

66.2

45

35

22.5

18.4

14

Construction of fusion CNS with various Construction of fusion CNS with various repeated EAAAK linkersrepeated EAAAK linkers

• CBP- (EAAAK)2 G-CNS

• CBP- (EAAAK)3 G-CNS

• CBP- (EAAAK)4 G-CNS

• CBP- (EAAAK)5 G-CNS

• CBP- (EAAAK)5 -CNS (Fusion protein without genenase I

cutting site)• (EAAAK)5 G-CNS (Fusion protein without CBP)

The fusion proteins were incubated in The fusion proteins were incubated in phosphate buffer (pH 6.0 at 16 phosphate buffer (pH 6.0 at 16 ℃℃) so that partial ) so that partial auto-cleavage fragments can be obtained. auto-cleavage fragments can be obtained.

SDS PAGE and MS analyses of auto-SDS PAGE and MS analyses of auto-cleavage of the fusion Proteincleavage of the fusion Protein

Lane 1: protein marker

Lane 2: CBP-V2G-CNS

Lane 3: CBP-V3G-CNS

Lane 4: CBP-V4G-CNS

Lane 5: CBP-V5G-CNS

Lane 6: CBP-V5-CNS

Lane 7: V5G-CNS

Lane 1: protein marker

Lane 2: CBP-V2G-CNS

Lane 3: CBP-V3G-CNS

Lane 4: CBP-V4G-CNS

Lane 5: CBP-V5G-CNS

Lane 6: CBP-V5-CNS

Lane 7: V5G-CNS

SDS PAGE and MS analyses of auto-SDS PAGE and MS analyses of auto-cleavage of the fusion Proteincleavage of the fusion Protein

Lane 1: protein marker

Lane 2: CBP-V2G-CNS

Lane 3: CBP-V3G-CNS

Lane 4: CBP-V4G-CNS

Lane 5: CBP-V5G-CNS

Lane 6: CBP-V5-CNS

Lane 7: V5G-CNS

SDS PAGE and MS analyses of auto-SDS PAGE and MS analyses of auto-cleavage of the fusion Proteincleavage of the fusion Protein

Lane 1: protein marker

Lane 2: CBP-V2G-CNS

Lane 3: CBP-V3G-CNS

Lane 4: CBP-V4G-CNS

Lane 5: CBP-V5G-CNS

Lane 6: CBP-V5-CNS

Lane 7: V5G-CNS

SDS PAGE and MS analyses of auto-SDS PAGE and MS analyses of auto-cleavage of the fusion Proteincleavage of the fusion Protein

Lane 1: protein marker

Lane 2: CBP-V2G-CNS

Lane 3: CBP-V3G-CNS

Lane 4: CBP-V4G-CNS

Lane 5: CBP-V5G-CNS

Lane 6: CBP-V5-CNS

Lane 7: V5G-CNS

SDS PAGE and MS analyses of auto-SDS PAGE and MS analyses of auto-cleavage of the fusion Proteincleavage of the fusion Protein

Lane 1: protein marker

Lane 2: CBP-V2G-CNS

Lane 3: CBP-V3G-CNS

Lane 4: CBP-V4G-CNS

Lane 5: CBP-V5G-CNS

Lane 6: CBP-V5-CNS

Lane 7: V5G-CNS

SDS PAGE and MS analyses of auto-SDS PAGE and MS analyses of auto-cleavage of the fusion Proteincleavage of the fusion Protein

Protocol of Protocol of one-pot one-pot protein protein

purificationpurification

CBP-V5G-LPHaseCBP-V5G-LPHaseCBP-V5G-CNSCBP-V5G-CNS

Lane 1: marker Lane 1: marker Lane 2: crude enzymeLane 2: crude enzymeLane 3: Lane 3: ββ-chitin purified enzyme-chitin purified enzymeLane 4: After auto-cleavage, the obtained target proteinLane 4: After auto-cleavage, the obtained target protein CNS: 24 kDa, LPHase: 40 kDaCNS: 24 kDa, LPHase: 40 kDa

Purification of His-Tagged Recombinant protein Purification of His-Tagged Recombinant protein using Nickel columnusing Nickel column

His-Tagged protein can bind to nickel column with moderate affinity and can be eluted with high concentration of imidazole.

His-Tag + auto-cleavage peptide + His-Tag + auto-cleavage peptide + magnetic particles magnetic particles

Will it work??Will it work??

One-step protein purification using MP and ACPOne-step protein purification using MP and ACP

12000 rpm ultra-sonication

12000 rpm

Target protein

Non-tagged protein

crude protein

His-tag binding

Wash & cleavage

remove MP

HisHis88--GFPGFP-(EAAAK)-(EAAAK)22--mcherrymcherry

10kD

17kD

28kD

35kD

48kD

63kD75kD

Lane 1: markerLane 2: crude enzymeLane 3: bound proteinLane 4: unbound protein after auto-cleavage

1 2 3 4

10kD

17kD

28kD

35kD

48kD

63kD75kD

100kD

Lane 1: markerLane 2: crude enzymeLane 3: MP bound with proteinLane 4: unbound protein after auto-cleavage

HisHis66-(EAAAK)-(EAAAK)33--GFPGFP 1 2 3 4

ConclusionsConclusions• The repeated EAAAK peptide exhibited an auto-cleavage The repeated EAAAK peptide exhibited an auto-cleavage

feature which can be mediated by pH condition. feature which can be mediated by pH condition. • With this system, many proteins have been successfully With this system, many proteins have been successfully

purified. purified.

• Integration of auto-cleavage peptide (ACP) technique with Integration of auto-cleavage peptide (ACP) technique with NTA-coated magnetic particles coated can simplify the NTA-coated magnetic particles coated can simplify the purification process.purification process.