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Protein Purification Involving a Unique Auto-Cleavage Feature of a Repeated EAAAK Peptide. Interdisciplinary Research of Enzyme Technology. Protein Production. Structure & Catalytic Mechanism. Bio-conversion. Enzyme Research. Bio-medical application. - PowerPoint PPT Presentation
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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.
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