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

1. Protein Purification Involving a Unique Auto-Cleavage Feature of a RepeatedEAAAK 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 Purification Techniques Protein Production 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 Transglycosylation Bio-medical application LC/MS/MS Biosensor/SPR SiNW-FET Nano-particles

4. Animal tissue Plant materials Microorganisms General Flow Chart of Purification Grinding Fermentation Extraction Extracellular enzymes Intracellular enzymes Disruption Filtration 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 Target protein Carrier Protein

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

9. Protein purification based on affinity binding Linker Binding Target protein Carrier 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 Linker • Costly affinity matrix required. Affinity matrix • Post proteolytic process needed.

14. Protein purification based on affinity binding Linker Binding Target protein Carrier 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 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 CBP Linker MCS Repeated EAAAK peptide with auto-cleavage property

16. 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. History of our finding…… Starting from the study of Chitinase from Bacillus NCTU2

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

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

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, pH6.0) 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 4.2 - 8.0 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 pH8.0 7.5 7.0 6.0 5.0 4.2 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 4.2 - 8.0 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. 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. 1 2 3 4 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 17kD 10kD

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.