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
Purification Techniques
Protein
Production
Structure &
Catalytic
Glycoside
hydrolases GH-1,Mechanism
Bio-conversion
Enzyme Research
Oligosaccharide
preparation
Biomass
degradation
Transglycosylation
GH-3, GH-17, GH-18,
GH-29, GH-46, GH-54,
GH-64, GH-72, GH-75
Bio-medical application
LC/MS/MS
Biosensor/SPR
SiNW-FET
Nano-particles
Purification
Animal tissue
Plant materials
Grinding
Extraction
Fermentation
Extracellular
enzymes
Filtration
Concentration
Microorganisms
Purification
Intracellular
enzymes
Disruption
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
Affinity matrix
Carrier Protein Target protein
Binding
Protein purification based on affinity binding
Binding
Affinity matrix
Linker
Carrier Protein
Target protein
• 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
Linker
Affinity matrix
• Costly affinity matrix required.
• Post proteolytic process needed.
Protein purification based on affinity binding
Binding
Affinity matrix
Linker
Carrier Protein
Target protein
• 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
Repeated EAAAK peptide
with auto-cleavage property
MCS
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
Catalytic
active ?
Linker
Chitin-binding domain
(1~150 aa)
Vector design
Linker
amino acid sequence
1
2
3
4
5
6
7
AKRGWI
GTGGEGPGG
GTGGEGPGGGGPGEGGTG
TTLGSNLGTLGLK
(EAAAK)5
GTGGEGPGGGGPGEGGTGGTGGEGPGG
(GGGGS)5
Nde1
PT7
CBP21 gene
Linker 1~7
protease cutting site
A
Procedure of pRSET/CBP-V5G vector construction
CBP
Linker
Protease cutting site
MCS
The chimeric chitinase is active
without significant improvement in
catalysis.
However, interestingly……
Linker
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?
M
pH8.0
7.5
7.0
6.0
5.0
4.2
66.2
45
CBP-V5G-CNS ( 45kDa)
35
22.5
18.4
100 ℃ for 10 min under pH 3.6；
exchange buffers with pH 4.2 8.0 and then kept at 25 ℃ for
12 h.
14
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 6.0 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 autocleavage 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 autocleavage 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 autocleavage 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 autocleavage 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 autocleavage 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
T a rge t pro tein
N o n -tag g e d pro tein
12 0 0 0 rp m
u ltra -so n icatio n
1 2 00 0 rp m
crud e p rote in
H is-tag b in din g
W ash & cle a vage
re m o v e M P
His8-GFP-(EAAAK)2-mcherry
1
75kD
63kD
48kD
35kD
28kD
17kD
10kD
2
3
4
Lane 1: marker
Lane 2: crude enzyme
Lane 3: bound protein
Lane 4: unbound protein after autocleavage
His6-(EAAAK)3-GFP
1
2
3
4
100kD
75kD
63kD
48kD
35kD
28kD
17kD
10kD
Lane 1: marker
Lane 2: crude enzyme
Lane 3: MP bound with protein
Lane 4: unbound protein after autocleavage
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.