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

2. Interdisciplinary Research of Enzyme Technology
Protein
Production
Structure &
Catalytic
Mechanism
Bio-conversion
Enzyme Research
Bio-medical application

3. Interdisciplinary Research of Enzyme Technology
Expression systems
Protein
Production
Purification Techniques
Structure &
Catalytic
Mechanism
Bio-conversion
Enzyme Research
Glycoside
hydrolases GH-1,
GH-3, GH-17, GH-18,
GH-29, GH-46, GH-54,
GH-64, GH-72, GH-75
Oligosaccharide
preparation
Biomass
degradation
What do we do with enzyme?
Transglycosylation
Bio-medical application
LC/MS/MS
Biosensor/SPR
SiNW-FET
Nano-particles

4. General Flow Chart of Purification
Animal tissue
Plant materials
Microorganisms
Grinding
Fermentation
Extraction
Extracellular
enzymes
Intracellular
enzymes
Disruption
Filtration
This slide shows you the general flow chart of protein purification. The protocol will be modified depending on the source of protein sample.
The sample need to be pretreated before applying for column chromatography. The process is complicated and time-consuming. Most of the time, the complete purification will require ion-exchange column, get filtration column, hydrophobic interaction column…and so on. Normally, it will take 1-2 weeks to
get tiny amount of the purified or partially purified protein.
Concentration
Purification
Pure Enzyme

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 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
Linker
Carrier Protein
Target protein
Affinity matrix
Binding

9. Protein purification based on affinity binding
Linker
Binding
Carrier Protein
Target protein
Affinity matrix
Glutathione S-transferase (Novagen, GST)
Maltose-binding protein (pMAL system, NEB)
Chitin-binding domain (IMPACT system, NEB)

10. Protein purification based on affinity binding
excess
wash

11. Dialysis to remove

12. Current strategies
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. Common Drawbacks Costly affinity matrix required. Linker
Post proteolytic process needed.

14. Protein purification based on affinity binding
Linker
Binding
Carrier Protein
Target protein
Affinity matrix
Glutathione S-transferase (Novagen, GST)
Maltose-binding protein (pMAL system, NEB)
Chitin-binding domain (IMPACT system, NEB)
Chitin-binding protein with auto-cleavage peptide linker
(developed by NCTU)

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

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

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

18. Vector design

19. Procedure of pRSET/CBP-V5G vector construction
MCS
CBP
Linker
Protease cutting site

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

21. The fusion protein broke into two fragments at pH 6.0!
CBP-V5G-ChiA (MW 58 kDa)
NCTU2 ChiA (36 kDa)
CBP (19 kDa)
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

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

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
66.2 45 35
22.5
18.4 14
CBP-V5G-CNS ( 45kDa)
100 ℃ for 10 min under pH 3.6；exchange buffers with pH and then kept at 25 ℃ for 12 h.
pH-dependent auto-cleavage of (EAAAK)5 linker!!

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
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. 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

29. 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

30. 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

31. 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

32. 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

33. Protocol of one-pot protein purification

34. CBP-V5G-CNS CBP-V5G-LPHase 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

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. His8-GFP-(EAAAK)2-mcherry

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

40. His6-(EAAAK)3-GFP 1 2 3 4 100kD 75kD 63kD 48kD 35kD 28kD
Lane 1: marker
Lane 2: crude enzyme
Lane 3: MP bound with protein
Lane 4: unbound protein after auto-cleavage

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