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

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

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

Strategy for massive production and purification of protein

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

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

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

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)

Protein purification based on affinity binding excess wash

Dialysis to remove

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.

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

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)

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

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.

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

Vector design

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

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!

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

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.

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

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. pH-dependent auto-cleavage of (EAAAK)5 linker!!

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)

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.

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

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

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

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

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

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

Protocol of one-pot protein purification

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

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.

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

One-step protein purification using MP and ACP

His8-GFP-(EAAAK)2-mcherry

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

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

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.