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

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Presentation on theme: "Protein Purification Involving a Unique Auto-Cleavage Feature of a Repeated EAAAK Peptide."— Presentation transcript:

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

2 Enzyme Research ProteinProduction Bio-conversion Bio-medical application Structure & CatalyticMechanism Interdisciplinary Research of Enzyme Technology

3 Enzyme Research ProteinProduction Bio-conversion Bio-medical application Structure & CatalyticMechanism Interdisciplinary Research of Enzyme Technology Expression systems Purification Techniques Oligosaccharide preparation Biomass degradation Transglycosylation LC/MS/MS Biosensor/SPR SiNW-FET Nano-particles Glycoside hydrolases GH-1, GH-3, GH-17, GH-18, GH-29, GH-46, GH-54, GH-64, GH-72, GH-75

4 General Flow Chart of Purification Animal tissue Plant materials Microorganisms ExtracellularenzymesIntracellularenzymes Disruption Filtration ConcentrationPurification Pure Enzyme Fermentation Extraction Grinding

5 Strategy for massive production and purification of protein

6 Current strategies and problems Recombinant protein technology is the best solution so far.

7 Current strategies and problems To simplify purification of recombinant proteins, several engineered affinity tags fusion protein can be purified to near homogeneity in a simple procedure. To simplify purification of recombinant proteins, several engineered affinity tags are used with which fusion protein can be purified to near homogeneity in a simple procedure. Linker Carrier Protein Target protein

8 Protein purification based on affinity binding Binding Linker Carrier Protein Affinity matrix Target protein

9 Protein 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) Binding Linker Carrier Protein Affinity matrix Target protein

10 Protein purification based on affinity binding wash excess

11 Dialysis to remove

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

13 Costly affinity matrix required. Costly affinity matrix required. Post proteolytic process needed. Post proteolytic process needed. Linker Affinity matrix Common Drawbacks

14 Protein 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 linker Chitin-binding protein with auto-cleavage peptide linker (developed by NCTU) (developed by NCTU) Binding Linker Carrier Protein Affinity matrix Target protein

15 A vector containing chitin-binding protein and repeated EAAAK peptide linker to form a simple and cost-effective system for protein expression and purification. A new system developed by our group MCS LinkerLinkerCBPCBP Repeated EAAAK peptide with auto-cleavage property

16 Characteristics of chitin-binding Protein (CBP) CBP promotes the hydrolysis of chitin catalyzed by chitinase. CBP promotes the hydrolysis of chitin catalyzed by chitinase. CBP has good binding specificity for chitin. CBP has good binding specificity for chitin. pH 8, CBP can bind to chitin. pH 8, CBP can bind to chitin. pH 6, CBP can be eluted. pH 6, CBP can be eluted. History of our finding…… Starting from the study of Chitinase from Bacillus NCTU2

17 Structures of Chitinases TIM-barrel structure Chitin-binding domain (1~150 aa) Linker CBP Catalytic active ? Bacillus NCTU2 ChiA Serratia ChiA

18 Vector design

19 Procedure of pRSET/CBP-V5G vector construction MCS CBPLinkerProtease cutting site

20 Linker CBP The chimeric chitinase is active without significant improvement in catalysis. However, interestingly…… However, interestingly…… The fusion protein broke into two fragments at pH 6.0!

21 Lane 1 Sample kept in water for hours (pH 6.9). Lane 2 Sample in Pi buffer (50 mM, pH 6.0) for 1 day Lane 3 Sample in Pi buffer (50, mM, pH 6.0) for 2 days Lane 4 Sample kept in water (pH 6.9) for 1 day Lane 5 Sample kept in water (pH 6.9) for 2 day CBP-V5G-ChiA (MW 58 kDa) NCTU2 ChiA (36 kDa) CBP (19 kDa) The fusion protein broke into two fragments at pH 6.0!

22 22.5 CBP-V5G-CNS( 45 kDa) Exchange buffers with pH and kept at 25 for 12 h. CBP (19 kDa) CNS (chitosanase, 24kDa) Other cases

23 Dose Auto-cleavage occur on CBP-(EAAAK) 5 -G-ChiA? Or contamination of protease?

24 pH-dependent auto-cleavage of (EAAAK) 5 linker!! M pH CBP-V5G-CNS ( 45kDa) 100 for 10 min under pH 3.6 exchange buffers with pH and then kept at 25 for 12 h

25 Construction of fusion CNS with various repeated 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)

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

27 SDS PAGE and MS analyses of auto-cleavage 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

28 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- cleavage of the fusion Protein

29 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- cleavage of the fusion Protein

30 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- cleavage of the fusion Protein

31 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- cleavage of the fusion Protein

32 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- cleavage of the fusion Protein

33 Protocol of one-pot protein purification

34 CBP-V5G-LPHaseCBP-V5G-CNS Lane 1: marker Lane 2: crude enzyme Lane 3: β-chitin purified enzyme Lane 4: After auto-cleavage, the obtained target protein CNS: 24 kDa, LPHase: 40 kDa CNS: 24 kDa, LPHase: 40 kDa

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

36 His-Tag + auto-cleavage peptide + magnetic particles Will it work??

37 One-step protein purification using MP and ACP

38 His 8 -GFP-(EAAAK) 2 -mcherry

39 10kD 17kD 28kD 35kD 48kD 63kD 75kD Lane 1: marker Lane 2: crude enzyme Lane 3: bound protein Lane 4: unbound protein after auto- cleavage

40 10kD 17kD 28kD 35kD 48kD 63kD 75kD 100kD Lane 1: marker Lane 2: crude enzyme Lane 3: MP bound with protein Lane 4: unbound protein after auto- cleavage His 6 -(EAAAK) 3 -GFP

41 Conclusions The repeated EAAAK peptide exhibited an auto-cleavage feature which can be mediated by pH condition. The repeated EAAAK peptide exhibited an auto-cleavage feature which can be mediated by pH condition. With this system, many proteins have been successfully purified. With this system, many proteins have been successfully purified. Integration of auto-cleavage peptide (ACP) technique with NTA-coated magnetic particles coated can simplify the purification process. Integration of auto-cleavage peptide (ACP) technique with NTA-coated magnetic particles coated can simplify the purification process.


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