Download Protein Folding and Expression

Protein Folding and Expression
Folding, Expression and Analysis
Contents
1. Introduction.......................................................................................................... 3
Contents
2. Expression Systems................................................................................................ 4
Human Cell-Free Expression System.......................................................................................................................................4
Brevibacillus Expression System II............................................................................................................................................6
B. subtilis Secretory Protein Expression System II...............................................................................................................8
3. Products for Increased Protein Yield and Purity..................................................... 9
pCold Expression Vectors.............................................................................................................................................................9
pCold TF Vector............................................................................................................................................................................ 10
pCold™ ProS2 DNA...................................................................................................................................................................... 10
mRNA Interferase™-MazF Enzyme......................................................................................................................................... 10
SPP System..................................................................................................................................................................................... 11
4. Mammalian Expression Vectors.............................................................................12
pBApo-CMV Vectors.................................................................................................................................................................... 12
pBApo-EF1a................................................................................................................................................................................... 12
pDON-AI-2....................................................................................................................................................................................... 13
pMEI-5.............................................................................................................................................................................................. 13
pDON-5............................................................................................................................................................................................ 14
5. Folding.................................................................................................................15
Chaperone Plasmid Set.............................................................................................................................................................. 15
Chaperonin GroE.......................................................................................................................................................................... 16
Corystein™ (Purothionin) Reagent......................................................................................................................................... 16
Refolding CA Kit............................................................................................................................................................................ 16
6. Application Notes.................................................................................................17
High-level Secretion of Recombinant Protein using the Brevibacillus Expression System.............................. 17
The pCold TF Protein Expression System Produces Soluble, Active Protein in E. coli........................................ 20
Unfolding the Potential of Proteins...................................................................................................................................... 22
7. FAQs.....................................................................................................................24
SPP System™ (Single Protein Production System)........................................................................................................... 24
pCold Expression Vectors.......................................................................................................................................................... 25
Refolding CA Kit............................................................................................................................................................................ 26
Chaperone Plasmid Set.............................................................................................................................................................. 27
8. Protein Sequencing and Analysis Products............................................................28
9. High Fidelity PCR Enzymes....................................................................................29
10.Takara Related Products.......................................................................................30
11.Clontech Related Products....................................................................................31
2
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
Introduction
Takara’s Protein Products
Protein Folding Products
Chaperone Assisted
Folding
Chaperonin GroE
Increase Level of Active Protein
Corystein™ Reagent
Reforming Disulfide Bonds
Chaperone Plasmid Set
Maximize Soluble Active Protein
Protein Expression Products
Insoluble Protein
or Protein Toxic to Cell
Refolding CA Kit
Optimize Refolding of
Inclusion Bodies
Increase Protein
Yield and Purity
pCold Vectors
Increase Recombinant Protein Yield
pCold TF DNA
Increase Expression using Trigger Factor
pCold Pro S2
Express Soluble Fusion Proteins
SPP System™ Kits
Single Protein Production System
Mammalian
Expression
pBApo-CMV Vectors
All-purpose Gene Expression Vectors
High Yield
Active Protein
Human Cell Free Expression System
High Efficiency Cell-free Protein Production
pBApo-EF1α Vectors
Brevibacillus Expression System II
pDON-AI-2 Vectors
B. subtilus Expression System
Strong promoter for increased expression
Retroviral Vector for High-efficiency
Gene Transduction
High Efficiency Protein Production
Secretory Protein Production
pMEI-5 Vectors
Retroviral Vector for High Expression
mRNA Interferase™ Plasmid
MazF Enzyme
pDON-5 Vector
Retroviral Vector for High-efficiency
Gene Transduction and High Expression
Active Protein
Ready for purification and/or analysis
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
3
1
Introduction
Protein expression is not an easy process; however, it can be made easier with the help of products that
aid expression and folding. There are many factors which can prohibit the production of functional protein.
Often it is not possible to predict which factors may impact expression of a particular protein of interest. In
order for production of your target protein to be successful, map the process out from the beginning. The
expression process consists of four steps: cloning, expression, purification, and characterization. Each step can
be optimized to obtain a sufficient amount of functional protein. Consider the end use of the protein. Is it for
biochemical studies or for structural analysis (NMR, X-ray crystallography)? The answer should help determine
the best production process for your needs. Takara Bio offers a wide selection of tools to aid every step of the
protein production process.
pT7-IRES His-N DNA
Human Cell-Free Expression System
Expression Systems
2
Code No. 3290
Shipping at － 20℃
Human Cell-Free Protein Expression System
3281
Size: 20 μg
Store at － 20℃
pT7-IRES His-N DNA
3290 pT7-IRES His-C DNA
3291 ＊ 2 years from date of receipt under proper storage
pT7-IRES Myc-N DNA
3292 conditions.
10 rxns
20 µg
20 µg
Usage :
20 µg
Protein expression using Human
Cell-Free Protein Expression System.
Features
Vector map for pT7-IRES His-N DNA :
Lot No.
Concentration
: as 1 h in a single-tube reaction
0.5 μg/μl
• Easy-to-use system allows generation
of protein in as little
40 μl
• Provides higher yield of functionalVolume
protein and: greater consistency than rabbit
reticulocyte or wheat germ in vitro translation systems
• Excellent with challenging proteins such as large proteins (over 150 kDa) and proteins
requiring post-translational modification
Regarding protocol for protein expression, please
• Amenable to high-throughput screening;
bulkthe
sizes
available.
refer to
product
manual for Human Cell-Free Pro-
Applications
tein Expression System (Cat. #3281).
Purity :
1． Confirmed to maintain the region from T7 promoter to T7 terminator
by dideoxy sequencing method.
2． Shown to be cleaved at a single site by Eco R I and at two sites by
Hin d III.
polyA
T7 terminator
Multiple Cloning Site
Factor Xa Site
His-Tag
EMCV
IRES
Ampr
pT7-IRES His-N DNA
(3,429 bp)
:
• Expression of toxic proteins that areDescription
lethal to host
cells of in vivo expression systems
pT7-IRES DNA series are expression vectors designed for Human Cell• Rapid analysis of protein function Free Protein Expression System. Tag sequence such as His-Tag or c-Myc
ColE1 ori
Tag, Factor Xa cleavage site, Multiple Cloning Site (MCS), polyA signal,
• Rapid analysis of mutation series orandtruncation
series:
quickly and
T7 terminator
are generate
located at protein
the downstream
of T7 promoter and
EMCV IRES. There are vectors with some arrangements for the kind of Tag
assess functionality using your downstream
assay
Figure 1. Map of pT7-IRES vector.
and the location.
• High-throughput proteomic studiespT7-IRES His-N DNA is an expression vector including His-Tag sequence.
Using pT7-IRES His-N DNA together with Human Cell-Free Protein
Expression
System
(Cat. #3281)
enables the expression
• Expression of proteins that are easily
degraded
or insoluble
in conventional
in vivo of your target
as N-terminal fusion of His-Tag. Factor Xa cleavage site is inserted
expression systems such as E. coli protein
so that His-Tag can be removed from the expressed fusion protein.
Note
pT7-IRES Vectors:
Target gene cloned into MCS in frame is transcribed as RNA-containingThis product is for research use only. It is not intended for use in
Each
vector
provides
20 or
µgdiagnostic
DNA at a procedures
concentration
of 0.5 µg/µL
(40 µL
total
therapeutic
for humans
or animals.
Also,
do volume).
EMCV IRES under the control of T7 promoter. By the effect of EMCV IRES,
Description
designed to promote protein translation initiation, efficient high level
not use this product as food, cosmetic, or household item, etc.
Takara products may not be resold or transferred, modified for resale
expression
be Takara
performed
The Human Cell-Free Protein Expression
Systemcan
from
BioinisHuman
easy toCell-Free
use. TheProtein Expression
transfer, or used to manufacture commercial products without
pT7-IRESor
Vector
Information
System.
written approval from TAKARA BIO INC.
single-tube reaction is easily assembled and protein synthesis is complete in as little as 1
If you require licenses for other use, please contact us by phone at
Vectors
in
the
pT7-IRES
series
T7 promoter
and EMCV IRES sequence to
Form
:
10 mM
Tris-HCl,
pH8.0
+81 77 543 7247 orinclude
from oura website
at www.takara-bio.com.
h at 32°C. The Human Cell-Free Protein
Expression
System
provides
high yield (e.g., Your use of this product is also subject to compliance with any
1 mM EDTA
facilitate
transcription
and
translation
in
the
Human
Protein
Expression System,
applicable licensing requirements describedCell-Free
on the product
web
50 µg/ml of human eIF4G) of functional protein, including proteins requiring
page. It isCloning
your responsibility
toconvenient
review, understand
and
adhere
to His-tag or Myc
as
well
as
a
Multiple
Site
(MCS),
tags
(Nor
C-terminal
Preparation
:
Purified
by
ion-exchange
column.
modifications such as glycosylation, phosphorylation, or fatty acylation. Excellent yield
any restrictions imposed by such statements.
tag sequences), a Factor Xa protease cleavage site for tag removal, poly-A
signal, and T7
is observed even with large proteins (over
kDa).
Genes
v201107Da
Chain150
length
: 3,249
bp of interest may be cloned
terminator.
rapidly into pT7-IRES vectors using In-Fusion cloning technology. After expression,
MCS :
proteins with N-terminal or C-terminal His-tag or N-terminal Myc tag can Nco
be generated,
I
depending on choice of vector. Bulk sizesEMCV
are available
for high-throughput studies;
IRES
Nhe I
5’- T AACGT・・・・・・T AA T A TGGCCACAACC ATG GC T AGC
contact [email protected] for more information.
• pT7-IRES His-N DNA (Cat. # 3290) encodes an N-terminal His-tag
Nde I
Sac I
Xho I
• pT7-IRES His-C DNAFactor
(Cat.Xa# 3291) encodes
a C-terminal
His-tag
CAC CA T CAC CA T CAC CA T AT C GAA GGG CGC CAT AT G GAG CT C CT C GAG
3’-A T TGCA・・・・・・A T T A T ACCGGTGT TGG T AC CGA T CG GTG GT•A GTG
GT A GTG
GT A
T AG
C T T# CCC
GT A T an
AC N-terminal
C T C GAG GAG
C T Ctag
pT7-IRES
Myc-N
DNA
(Cat.
3292)GCG
encodes
c-Myc
MetforAla
In vitro translation has many advantages for protein expression: it is excellent
rapidSer His His His His His His Ile Glu Gly Arg His Met Glu Leu Leu Glu
c II
Storage
studies of protein function or features, amenable to high-throughputHinstudies,
useful for
Bam H I
Eco R I
Spe I
Pst I
Xba I
End
Sal I
proteins that are degraded or insoluble
with
in
vivo
systems,
and
can
be
used
to
generate
Cell-Free Protein Expression System (Cat. # 3281): –80°C
GT AA T C-3’
GGA T CC GAA T T C AC T AGT GT C GAC C TG CAG T C T AGA T•AGHuman
lethal proteins that cannot be expressed
systems
toxicity.
In contrast
CC Tusing
AGG in
C Tvivo
T AAG
TGA Tdue
CA to
CAG
C TG GAC
GT C AGA T C T A T C CA T T AG-5’
•
pT7-IRES
Vectors (Cat. #s 3290, 3291, 3292): –20°C
Gly inSer
Phe Thr systems
Ser Valalso
Asp
Leu postGln Ser Arg
to expression using prokaryotic host cells,
vitroGlutranslation
allow
translational modifications such as glycosylation, phosphorylation, and fatty acylation.
Related In-Fusion Cloning Products
Kit Components (for 10 × 20-μL reactions)
(1) Cell Lysate*1 100 μL
(2) Mixture-1 60 μL
(3) Mixture-2*2 10 μL
(4) Mixture-3*2 20 μL
(5) T7 RNA Polymerase (200 U/μL) 10 μL
(6) pT7-IRES Vector (0.5 μg/μL) 20 μL
(7) Control Vector*3 (0.3 μg/μL) 5 μL
*1: Dissolve just prior to use; gently and thoroughly mix with a micropipette and
use immediately. After use, promptly store at –80°C.
Note: Although five cycles of freeze-thaw generally would not lead to any
decline in performance, the cell lysate should be stored in aliquots of the
required volume.
*2: Mixture-2 and Mixture-3 contain protein. To avoid protein deactivation, do not
stir excessively or vortex. Mixture-2 contains an HN-tagged protein.
*3: This vector harbors a β-galactosidase gene.
4
His-Tag
For rapid and easy cloning, use the In-Fusion HD Cloning System (Clontech Cat. #
639645/ 639646/639692/ 639647) or In-Fusion HD Cloning System CE (Clontech Cat. #
639636/ 639637/639693/ 639638) to generate pT7-IRES constructs with your insert of
interest.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
2
Expression Systems
Figure 2. Principle of the Human Cell-Free Protein Expression System.
A) In an easy and simple protocol, target protein translation is initiated by adding pT7-IRES Vector containing the target gene cassette and other kit components. B) The target gene RNA transcribed from
the pT7-IRES Vector has an IRES sequence designed to promote protein translation initiation. As protein synthesis progresses, the translation initiation factor from the cell lysate becomes inactivated. The
translation enhancement factor in the reaction mixture, however, reactivates this inactivated translation initiation factor and thereby maintains a high level of translation.
Relative absorbance
120
1
2
3
100
80
Mixture-2(＋)
Mixture-2(ー)
60
40
20
0
0
1
2
3
4
5
6
7
8
◄◄
Time (hour)
Figure 3. Time course of β-galactosidase expression and effect
of translation enhancement factor.
Eight replicates of β-galactosidase in vitro translation were performed using 1 µL of
Control Vector per reaction. At each of the indicated time points (0, 0.5, 1, 2, 3, 4.5,
6, and 8 h after start of the reaction), one reaction tube was removed and used for
β-galactosidase activity assay with O-nitrophenyl-β-D-galactopyranoside (ONPG) as
the substrate. A separate set of reactions were conducted in absence of the translation
enhancement factor (Mixture-2). The activity of β-galactosidase increased over time
to peak at approximately 4.5 h. Additionally, the presence of Mixture-2 containing
translation enhancement factor markedly increased yield of active protein.
Figure 4. Synthesis of high molecular weight
proteins using the Human Cell-Free Protein
Expression System.
In vitro translation reactions were performed to synthesize human
Dicer (200 kDa, lane 2) or human eIF4G (170 kDa, lane 3) protein.
Reactions were analyzed by SDS-PAGE and Coomassie blue staining.
Arrowheads indicate target proteins. Lane 1, negative control.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
5
Brevibacillus Expression System II
Expression Systems
2
Brevibacillus Expression System II
Brevibacillus choshinensis Competent Cells pNC-HisE DNA
pNC-HisF DNA
pNC-HisT DNA
pNCMO2 DNA
pNY326-BLA DNA
pNI DNA
pNI-His DNA
pNY326 DNA
HB200
HB116
HB123
HB122
HB121
HB112
HB114
HB131
HB132
HB111
1 Kit
100 µL x 10
10 µg
10 µg
10 µg
10 µg
1 µg
10 µg
10 µg
10 µg
Features
• Efficient production of secreted or intracellular target proteins
• Produces negligible amounts of extracellular protease – products remain intact in
culture medium
• Unlike E. coli, produces no endotoxins
• Proteins are produced in active form
• Easy to culture, handle, and sterilize
Description
Brevibacillus choshinensis is a gram-positive bacterium with exceptional capacity for
heterologous protein expression.
The Brevibacillus Expression System II enables highly efficient production of target
protein in secreted form. This system allows high yield of active proteins and is wellsuited for expression of eukaryotic proteins. The Brevibacillus system is nearly free of
proteases, which facilitates production of intact protein products.
Examples of successfully expressed proteins can be seen in Table 1. This includes
expression of enzymes, antigens, and cytokines. Each protein was produced at a very
high level of expression and confirmed to have native biological activity. In addition,
proteins from taxonomically distant organisms were successfully produced, such as
eubacteria, archaebacteria, eukaryotes, and viruses.
The Brevibacillus system facilitates disulfide bond formation (commonly required in
proteins of eukaryotic origin). In addition, B. choshinensis serves as an excellent host for
intracellular protein production, frequently producing intracellular proteins in soluble
form in the cytoplasm without forming inclusion bodies. The Brevibacillus system often
works better than E. coli for expression of particular targets.
Utilizing His-tag containing vectors (pNC-HisE, pNC-HisF, pNC-HisT, pNI-His) allows
effective purification of the expressed target protein. Tags can be removed by protease
treatment following purification.
6
Proteins
Enzymes
α-amylase
α-amylase
Sphingomyelinase
Sphingomyelinase
Xylanase
Xylanase
CGTase
CGTase
Chitosanase
Chitosanase
Hyper
Hyperthermo-stable
thermo-stableprotease
protease
Hyper
Hyperthermo-stable
thermo-stablenuclease
nuclease
PDI
PDI
Antigens
Surface
Surfaceantigen
antigen
Surface
Surfaceantigen
antigen
Cytokines
EGF
EGF
IL-2
IL-2
NGF
NGF
IFNIFN-γ γ
TNF-α
TNF-α
GM-CSF
GM-CSF
GH
GH
Origins
Origins
Production（
Production（
g/L ） g/L
B. licheniformis
B. cereus
B. halodurans
B. macerans
B. circulans
A. pernix
P. horikoshii
human
3.7
3.0
3
0.2
1.5
1.4
0.1
0.7
1.0
1
E. rhusiopathiae
T. pallidum
0.90.9
0.80.8
human
human
mouse
chicken
bovine
bovine
flounder
1.5
0.6
0.2
0.5
0.4
0.2
0.2
Table 1: Example of Proteins Expressed using the Brevibacillus
Expression System
System Components:
Product Name
Brevibacillus Expression System II
Kit Components
Expression Vectors
pNY326A DNA
pNCMO2 DNA
Control Vector
pNY326-BLA DNA
Competent Cells
Brevibacillus choschinesis Competent cells
MT Medium
Solu on A
Solu on B
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
Catalog #
HB200
Quanty
Kit
HB111
HB112
10 µg
10 µg
HB114
1 µg
HB116
100 µL x 10 tubes
1 ml x 10
1 ml
1 ml x 2
Vectors for the Brevibacillus System
Lac operator
P2 promoter
sec signal peptide
pNC-HisE
(5,263 bp)
pNC-HisF
(5,260 bp)
rep
＋
ColE1 ori
(5.2 kb）
r
rep
ori +
2
rep
Ampr
P5 promoter
sec signal peptide
multiple cloning site
X-terminator
ori +
pNY326
(3.4 kp)
Nm
pNI DNA
(5,055 bp)
rep
Nmr
r
multi cloning site
ColE1 ori
pNI-His DNA
(5,079 bp)
rep
ori –
Lac operator
ori +
NmR
All shuttle vectors between B. choshinensis and
E. coli contain the P2 promoter, which is one of
the five promoters that control transcription of
the cell wall protein gene (HWP). This promoter
functions only in B. choshinensis and not in E. coli,
thereby ensuring robust protein production only in
B. choshinensis.
ori –
Ampr
NmR
ori
P2 promoter
rep
Nm r
Expression Systems
NmR
pNCMO2
pNC-HisT
(5,260 bp)
Lac operator
His-Tag
multi cloning site
ColE1 ori
AmpR
AmpR
ori –
ori –
ori +
ColE1 ori
rep
P2 promoter
His-Tag
MCS
ori –
ori＋
ColE1 ori
AmpR
Amp
His-Tag
MCS
ori−
ori＋
ColE1 ori
Lac operator
P2 promoter
sec signal peptide
Lac operator
P2 promoter
sec signal peptide
His-Tag
MCS
ori−
Nmr
The pNY326 vector* is maintained more stably than pNC or pNI
vectors in the host cells due to much weaker promoter activity and
smaller size (3.4 kb). The host strains containing the vector can be
repeatedly subcultured, may be used for scaled-up production,
and will continue to stably produce protein. The pNY326 vector
must be constructed by a one-step method using B. choshinensis.
Brevibacillus choshinensis Competent Cells are used as the
transformation host.
*Can only be maintained in B. choshinensis
Choosing a Brevibacillus Vector?
His-Tag
Sec Signal Peptide
Construct in
Protease
cleavage site
X-terminator
Yes
Yes
Yes
E. coli
Enterokinase
No
Secretory
Yes
Yes
Yes
E. coli
Thrombin
No
Shuttle Vector
Secretory
Yes
Yes
Yes
E. coli
Factor Xa
No
Shuttle Vector
Intracellular
Yes
Yes
No
E. coli
Enterokinase
No
Shuttle Vector
Intracellular
Yes
No
No
E. coli
No
No
Expression
Secretory
No
No
Yes
Brevibacillus
No
Yes
pNCMO2 (5.2 kb)
Shuttle Vector
Secretory
No
No
Yes
E. coli
No
Yes
pNY326-BLA
Positive Control
Secretory
Vector Name
Vector Type
pNC-HisE (5,263 bp)
Shuttle Vector
Secretory
pNC-HisT (5,260 bp)
Shuttle Vector
pNC-HisF (5,260 bp)
pNI-His DNA (5,079 bp)
pNI DNA (5,055 bp)
pNY326 (3.4 Kb)
Expression Vector Lac Operator
Includes a gene encoding Bacillus licheniformis a-amylase (55 kDa)
Clontech offers a broad range of products for purifying His-tagged proteins. Please see the Clontech Related Products section on page (31) for ordering information.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
7
B. subtilis Secretory Protein Expression System
B. subtilis Secretory Protein Expression System
3380 10 rxns
Features
Description
Bacillus subtilis has become an increasingly popular host for recombinant protein
expression. With its ability to secrete protein directly into culture media, amenability to
medium- and large-scale fermentation, lack of codon bias, and designation by the U.S.
Food and Drug Administration as an organism that is Generally Regarded As Safe (GRAS),
it’s no wonder that the majority of industrially-produced enzymes are expressed in
Bacillus species such as B. subtilis. Optimization of secretion, however, can be necessary
to achieve highest yields. To address this, the B. subtilis Secretory Protein Expression
System from Takara Bio allows rapid development of a library of B. subtilis clones, each
bearing a pBE-S construct in which the ORF for your protein of interest is fused with
sequences for 173 unique signal peptides. Perform a downstream assay to identify and
select clones which secrete the highest amount of functional protein into the culture
media, and you can quickly identify the signal peptide that results in efficient expression
of your desired secreted protein.
multi cloning site (MCS)
His-Tag
pBE-S DNA
(5,938 bp)
Kanr
ColE1
Ampr
Figure 2. Vector map for pBE-S DNA, a B. subtilis/E. coli shuttle vector
used with the B. subtilis Secretory Protein Expression System.
Kit Components*1
SP DNA mixture (0.032 pmol/μL)*2 45 μL
pBE-S DNA (0.5 μg/μL)*3 20 μg
100 μL x 2
B. subtilis RIK1285*4 (glycerol stock) *1: Library development (10 reactions)
*2: DNA mixture encoding secretory signal peptides from the 173 types of B. subtilis
for use with In-Fusion cloning system (10 reactions). For the sequence of secretory signal peptides, please see the product page for the B. subtilis Secretory
Protein Expression System on the Takara Bio web site.
*3: in TE buffer (pH 8.0)
*4: Marburg 168 derivative: trpC2 , lys1 , aprE Δ3, nprR2 , nprE18
The System provides sufficient reagents for 10 library development reactions.
Storage
• SP DNA mixture and pBE-S DNA: –20°C
• B. subtilis RIK1285 glycerol stock: –80°C
A405
• Expression of soluble, recombinant protein secreted directly into the culture media
• Protein expression in a host amenable to medium- and large-scale fermentation in
addition to small-scale culturing
• Expression of proteins with complex structure, such as proteins with disulfide (S-S)
bonds
• Generation of target protein in a host that is considered to be Generally Regarded As
Safe (GRAS) by the U.S. Food and Drug Administration
• Useful for producing easily purified recombinant protein – with proper in-frame
cloning, a C-terminal His-tag can aid purification from culture media
52 I
SP
A405
Applications
promoter
pUB
A405
Expression Systems
2
173 different types of SP DNA
are inserted into this region
in place of the
SP
I
• Includes pBE-S DNA, an E. coli/B. subtilis shuttle vector with B. subtilis-derived
subtilisin (aprE) promoter, secretory signal peptide (aprE SP), Multiple Cloning Site,
and 3’ (C-terminal) His-tag sequence
• Supplied with SP DNA Mixture, a library of DNA sequences encoding 173 unique
secretory signal peptides that can be inserted upstream of your target gene
• Fully compatible with In-Fusion cloning kits and systems to allow rapid and easy
construct generation
• Includes B. subtilis strain RIK1285
3
2.5
2
1.5
1
0.5
0
1
3
2.5
2
1.5
1
0.5
0
161
3
2.5
2
1.5
1
0.5
0
321
11
21
31
41
51
61
71
81
91
101
111
121
131
141
151
◄
171
181
191
201
211
221
231
241
251
261
271
281
291
301
311
◄
331
341
351
361
371
381
391
401
411
421
431
441
451
461
◄
䠝㻮
Figure 3. Results of measuring β-glycosidase activity of 470 clones of
an expression library with different signal peptide sequences.
Clones showing activity levels of varying strengths were observed. The arrowheads indicate the
expression level observed with the aprE signal peptide.
Related In-Fusion Cloning Products
For rapid and easy cloning, use the In-Fusion HD Cloning System* (Clontech Cat. #
639645/639646/639692/639647) or In-Fusion HD Cloning System CE* (Clontech Cat.
# 639636/639637/639693/639638) to generate pBE-S constructs with your insert of
interest.
Figure 1. Flowchart of the experimental procedure for
the B. subtilis Secretory Protein Expression System.
8
*: Available in the U.S. only. Outside of the U.S., use the In-Fusion HD Cloning Kit (Clontech Cat.
# 639648/639649/639650) or In-Fusion HD Cloning Kit w/ Cloning Enhancer (Clontech Cat. #
639633/639634/639635) in combination with high-efficiency Stellar Competent Cells (Clontech
Cat. # 636763/636766), a HST08 E. coli strain. Availability of In-Fusion systems and kits varies by
geographic location; check for products sold in your region.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
pCold Expression Vectors
pCold Vector Set
pCold I DNA
pCold II DNA
pCold III DNA
pCold IV DNA
3360
3361
3362
3363
3364
Features
1 Set (ea. 5 µg)
25 µg
25 µg
25 µg
25 µg
Description
Related Products
Takara’s pCold Expression Vectors offer Cold Shock expression technology for high purity,
high yield protein production.
Chaperone Plasmid Set, 3340, p. 15.
The pCold series includes four different vectors. Each includes the Cold Shock Protein
A (cspA) promoter for expression of highly pure recombinant protein in E. coli at high
yield. These vectors selectively induce target protein synthesis at low temperature
Reference
• Great for difficult proteins that can’t be expressed with the T7 system
• Facilitates increased solubility due to expression at reduced temperature
• Facilitates increased purity due to repressed expression of host proteins
Application
1. Quing, G., et. al. (2004) Nature Biotechnol. 22(7):877-882.
Takara’s pCold expression vectors
pCold I
M
pCold II
cspA 3'UTR
multiple cloning site
cspA 5'UTR
lac operator
cspA promoter
M
4.4kb
IG
13
pCold IV
Amp
ColE1 ori
ColE1 ori
IG
13
pCold III
Amp
4.4kb
Amp
Amp
4.4kb
IG
13
cspA 3'UTR
multiple cloning site
TEE
cspA 5'UTR
lac operator
cspA promoter
lacI
M
lacI
IG
13
lacI
M
cspA 3'UTR
multiple cloning site
His•Tag
TEE
cspA 5'UTR
lac operator
cspA promoter
ColE1 ori
4.4kb
lacI
cspA 3'UTR
multiple cloning site
Factor Xa site
His•Tag
TEE
cspA 5'UTR
lac operator
cspA promoter
ColE1 ori
In the following examples, genes that were poorly expressed or that produced insoluble protein with the T7 promoter expression system were expressed using the pCold system. pCold I DNA was used as
an expression vector in E. coli. Expression from T7 promoter-driven vectors was induced with IPTG and T7 plasmid-containing cells were cultured at 37°C.
N.C T7 pCold
T7
kDa
T
S
pCold
T
S
kDa
T: Entire protein fraction
S: Soluble fraction
97.4
97.4
66.2
← Expression level increased
66.2
45
45
31
← Expression enabled
21.5
21.5
14.4
14.4
CBB staining of the entire protein fraction
Figure 1. Expression of human gene A.
31
Human gene A (~31 kDa) was expressed in both the T7 system and the cold-shock expression
system. No expression was observed in the T7 system, but human gene A was expressed in the
pCold system.
CBB staining
Figure 2. Expression of human gene C.
Comparison of expression of soluble human gene C protein (~80 kDa) in the cold-shock
expression system vs. the T7 system was performed. Target protein in the soluble fraction of
pCold cells was dramatically higher than that of the T7 system.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
9
3
Products for Increased Protein Yield and Purity
• High-efficiency protein expression using a cold shock promoter
(15°C), a condition at which host protein synthesis is suppressed and protease activity is
decreased. This results in high yields of target protein (~60% of intracellular protein).
In addition to the cspA promoter, all four vectors contain a lac operator (for control
of expression), ampicillin resistance gene (amp r ), ColE1 origin of replication, M13 IG
fragment, and multiple cloning site (MCS). Three vectors also contain either a translation
enhancing element (TEE), His-Tag sequence, and/or Factor Xa cleavage site. These
vectors work equally well for synthesis of non-labeled and radiolabeled proteins and can
be used with Takara’s Chaperone Plasmid Set (Cat. # 3340).
pCold TF Vector
pCold TF DNA
3365
25 µg
Application
3
• Highly efficient protein expression using cold shock technology
• High yield of active protein due to Trigger Factor chaperone as a solubility-promoting
fusion tag
kDa
pCold
1 2
pCold +
Chaperone
1 2
T7
1 2
1. Cell extract solution
2. Soluble fraction
target protein
97
Description
Products for Increased Protein Yield and Purity
pCold TF
1 2
66
Takara’s pCold TF DNA Vector is a fusion cold shock expression vector that expresses
Trigger Factor (TF) chaperone as a soluble fusion tag. Trigger Factor is a 45 kDa
prokaryotic ribosome-associated chaperone protein that facilitates co-translational
folding of nascent polypeptides. Because of its E. coli origin, TF is highly expressed
in E. coli expression systems. The pCold TF DNA Vector consists of the cspA promoter
plus additional downstream sequences including a 5’ untranslated region (5’ UTR), a
translation enhancing element (TEE), a His-Tag sequence, and a multiple cloning site
(MCS). A lac operator is inserted downstream of the cspA promoter to ensure strict
regulation of expression. Additionally, recognition sites for HRV 3C Protease, Thrombin,
and Factor Xa are between TF-Taq and the Multiple Cloning Site (MCS). These sequences
facilitate tag removal from the expressed fusion protein. Most E. coli strains can serve as
expression hosts.
45
* co-expressed
trigger factor
*
31
22
Figure 1. Expression of protein A in T7 and pCold systems
The expression of enzyme protein A (~29 kDa) was poor when utilizing aT7 or pCold I expression
systems, even when the pCold I construct was co-expressed with a chaperone. In contrast, the
expression of target protein as a fusion (29 kDa + 52 kDa) was successful with pCold TF DNA,
and most of the expressed protein was in soluble form. The expressed enzyme protein A showed
activity even in the form of a fusion protein (data not shown).
pCold ProS2 DNA
pCold™ ProS2 DNA
3371
25 µg
Features
Description
• Facilitates high yield protein expression with optimized protein folding
• Enables expression of fusion proteins with a soluble tag to optimize solubility
The pCold Pro S2 expression vector features Protein S, a soluble tag from Myxococcus
xanthus fused to the N-terminus of target proteins. Tight regulation of protein expression
is maintained by a lac operator downstream of the cold shock promoter. HRV 3C
Protease, Thrombin, and Factor Xa recognition sites are encoded between the Protein S
tag and the MSC to facilitate tag removal.
mRNA Interferase™-MazF Enzyme
mRNA Interferase™-MazF 2415A
1000 units
Application
Description
• Site-dependent cleavage of ssRNA
MazF is a toxin protein in the toxin-antitoxin module of E. coli. It possesses
endoribonuclease activity and specifically cleaves single-stranded RNA at the 5’ end
of 5’-ACA-3’ sequences. This enzyme does not cleave double-stranded RNA, doublestranded DNA, or single-stranded DNA. mRNA Interferase-MazF is supplied as a fusion
protein of E. coli MazF and Trigger Factor. The Trigger Factor protein is an E. coli
chaperone protein. The enzyme is also supplied with a 5X MazF buffer (200 mM Sodium
phosphate, pH 7.5, 0.05% Tween 20.)
10
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
SPP System
SPP System™ I
SPP System™ II
SPP System™ III
SPP System™ IV
SPP System™ I-IV
3367
3368
3369
3370
3366
1 kit
1 kit
1 kit
1 kit
1 kit
3
Application
• P referential expression of target protein by suppression of endogenous proteins
using mRNA interferase plasmid
Products for Increased Protein Yield and Purity
Description
This system utilizes an E. coli protein, MazF, described as an mRNA interferase by Suzuki
et. al. MazF is a sequence-specific endoribonuclease that cleaves single strand RNAs at
5’-ACA-3’ (ACA) sequences. When using the SPP System, the transcript of interest should
therefore lack ACA sequences. MazF is co-expressed in the E. coli host and suppresses
expression of non-target genes by cleaving host transcripts at ACA sequences. Therefore,
the target protein is the most abundantly expressed protein (Figure 1). Because of the
requirement for target gene transcripts to lack ACA sequences, the SPP System is not
suitable for all genes of interest; however, when appropriate, it can result in extremely
high levels of protein production.
Reference
Suzuki, M., et. al. (2005) Molecular Cell 18(2)253-261.
Figure 1. Synthesis of cspA-promoter expressed envAB in
presence and absence of MazF.
E. coli BL21 cells co-expressing MazF and pCold (SP-4) envZB showed good envZB
expression and extremely low background synthesis of host proteins.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
11
pBApo-CMV Vectors
pBApo-CMV Neo DNA pBApo-CMV Pur DNA pBApo-CMV DNA 20 µg
20 µg
20 µg
Features
• General purpose gene expression vector for mammalian cells
• Can be used to express miRNA precursors and other transcription products
• Allows easy transfer of expression cassette to adenovirus vector
EcoR V
Cla I
EcoR V
EcoR I
SV40 polyA
BamH I Xba I
Description
Sse8387 I
HSV TK polyA
EcoR V
pBApo-CMV is a general purpose gene expression vector for mammalian cells. This
vector has a promoter from cytomegalovirus (CMV IE promoter), a poly(A) signal from
thymidine kinase of herpes simplex virus (HSV), and a multiple cloning site (MCS). This
vector can be used to express miRNA precursors and other transcription products in
addition to protein-coding genes. The cassette promoter + ORF + poly(A) signal cassette
can be easily transferred from this vector to an adenovirus vector.
Sph I
HSV TK polyA
Cla I
or
BamH I
Xba I Sal I
Acc I
Hinc II
Pst I/Sse8387 I
CMV IE promoter
Hind III
pBApo-CMV Neo
EcoR I
Cla I
CMV IE promoter
NeoR
QT
PurR
Hind III
EcoR V
pBApo-CMV Pur
Cla I
pBApo-CMV
SV40 promoter
AmpR
AmpR
With the ability to achieve a high infection efficiency across a broad spectrum of cell lines,
adenovirus vectors are suitable for in vitro and in vivo gene transduction. For constructing
recombinant adenoviruses, use Adenovirus Expression Vector Kit (Dual Version) Ver. 2
(Cat. # 6170).
In addition to a basic vector (Cat. # 3242), the pBApo-CMV series also includes vectors
with a neomycin resistance cassette (Cat. # 3240) or a puromycin resistance cassette (Cat.
# 3241) for stable expression in mammalian cells.
Figure 1: Expression of DsRed-Express with pBApo-CMV Neo.
HEK 293 cells were transfected with a pBApo-CMV Neo construct bearing a DsRed-Express
cassette. The cells were visualized by fluorescence microscopy 2 days after transfection.
pBApo-EF1alpha
pBApo-EF1alpha Neo DNA
pBApo-EF1alpha Pur DNA 3243
3244
20 µg
20 µg
Features
RV
RV
I
• EF1 alpha promoter directs higher level of expression than CMV IE promoter
• Can be used to express miRNA precursors and other transcription products
• Allows easy transfer of expression cassette to adenovirus vector
Application
I
RI
HI
RI
EF1α promoter
SV40 polyA
EF1α promoter
SV40 polyA
I
NeoR
HI
8387 I
I
PurR
d III
d III
RV
RV
I
pBApo-EF1α Neo
5,167 bp
SV40 promoter
8387 I
HSV TK polyA
HSV TK polyA
pBApo-EF1α Pur
I
4,972 bp
SV40 promoter
• Gene expression in mammalian cells
AmpR
AmpR
Description
Transient expression
%positive
70
300
60
250
20
60
35
50
EFp
CMVp
150
EFp
CMVp
100
25
20
15
40
EFp
CMVp
MFI
200
% positive
30
MFI
40
30
50
40
Stable Expression
%positive
MFI
MFI
The pBApo-EF1alpha series includes simple gene expression vectors for mammalian
cells. These vectors include a promoter from human polypeptide chain elongation factor
(EF-1 alpha promoter) and a poly(A) signal site from herpes simplex virus thymidine
kinase gene. They can also be used to express miRNA precursors and other transcripts
in addition to protein-encoding genes. Versions with neomycin or puromycin resistance
cassettes are available. A promoter + ORF + poly(A) signal cassette can be easily
removed from these vectors and transferred to an adenovirus vector. Adenovirus vectors,
which have high infection efficiency and a wide target cell spectrum, are suitable for in
vitro and in vivo gene transductions. For constructing recombinant adenoviruses, the
Adenovirus Expression Vector Kit (Dual Version) Ver. 2 (Cat. # 6170) is recommended.
% positive
Mammalian Expression Vectors
4
3240
3241
3242
30
EFp
CMVp
20
10
10
10
50
0
0
0
0
ES-E14TG2a
ES-E14TG2a
ES-E14TG2a
ES-E14TG2a
5
Figure 2: Expression levels of pBApo-EF1α Neo or pBApo-CMV Neo.
Expression of AcGFP1 was assessed in mouse ES cells transiently or stably expressing
the target gene under the direction of either the EF1α promoter or CMV IE promoter.
12
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
pDON-AI-2
pDON-AI-2 DNA pDON-AI-2 Neo DNA 3654
3653
20 µg
20 µg
Features
5' LTR
Amp
HCMV
IE promoter
MLV R
MLV U5
r
PmaCI (1261)
Apa I (1266)
Sac II (1270)
Not I (1272)
Bgl II (1280)
Cla I (1286)
Bam HI (1292)
Sal I (1298)
Hpa I (1304)
SD
ψ
pDON-AI-2
Intron + SA
4586 bp
MLV U3
Amp
HCMV
IE promoter
MLV R MLV U5
r
PmaCI (1261)
Apa I (1266)
Sac II (1270)
Not I (1272)
Bgl II (1280)
Cla I (1286)
Bam HI (1292)
Sal I (1298)
Hpa I (1304)
SD
ψ
Intron + SA
pDON-AI-2 Neo
Minimal
SV40 promoter
5719 bp
r
Neo
MLV R
MLV U5
MLV U5
MLV U3
MLV R
3' LTR
Application
3' LTR
• Retrovirus-mediated gene transfer into mammalian cells
Description
The retroviral vector pDON-AI-2 and pDON-AI-2-Neo do not contain any MoMLV
derived genes (gag, pol, or env coding sequences) except LTR and packaging signal
(psi sequence). The U3 region of 5’ LTR has been substituted with a stronger promoter
derived from cytomegalovirus, giving these vectors a high transcription efficiency
and allowing them to be used to generate high titer-recombinant retroviruses and
accordingly efficient gene transductions. Moreover, they carry a human actin-derived
intron and splice acceptor upstream of the cloning site to increase the efficiency of
target gene expression after gene transduction. pDON-AI-2 Neo DNA (Cat.# 3653) has a
neomycin resistance gene as a drug selection marker.
pMEI-5
pMEI-5 DNA
pMEI-5 Neo DNA
3656
3655
20 µg
20 µg
Features
• For highly efficient transcription
• To increase target gene expression after gene transduction, includes a human EF1αderived intron with high splicing activity upstream of the cloning site
Application
Amp
Amp
r
MLV U3
MLV U5
pMEI-5
The retroviral vectors pMEI-5 and pMEI-5-Neo possess LTR and psi (viral packaging
signal), but lack the structural genes necessary for particle formation and replication
(gag, pol, and env). Because these vectors contain a human EF1α-derived intron with
high splicing capacity, they facilitate high transcription efficiency. pMEI-5 Neo DNA
SD
ψ
4689 bp
MLV U5
MLV U3
MLV R
3' LTR
Description
5' LTR
MLV U3
MLV R
MLV U5
MLV R
Intron + SA
• Retrovirus-mediated gene transfer into mammalian cells
r
5' LTR
MCS
PmaCI (1760)
Apa I (1765)
Sac II (1769)
Not I (1771)
Bgl II (1779)
Cla I (1785)
Bam HI (1791)
Sal I (1797)
Hpa I (1803)
Xho I (1811)
5820 bp
MLV U5
MLV R
MLV U3
3' LTR
Intron + SA
minimal
SV40 promoter
Neo
MCS
SD
ψ
pMEI-5 Neo
PmaCI (1760)
Apa I (1765)
Sac II (1769)
Not I (1771)
Bgl II (1779)
Cla I (1785)
Bam HI (1791)
Sal I (1797)
Hpa I (1803)
r
contains the neomycin resistance gene as a selective marker. Gene expression levels 2- to 8-fold higher than pDON-Al-2 series can be expected.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
4
13
Mammalian Expression Vectors
• Enables highly efficient gene transduction
• Retroviral vector that lacks all MoMLV-derived genes (gag , pol , or env) except LTR
and packaging signal (Ψ sequence)
• Includes a human actin-derived intron and splice acceptor for efficient target gene
expression
MCS
5' LTR
MCS
pDON-5
pDON-5 DNA
pDON-5 Neo DNA 4
3658
3657
20 µg
20 µg
5' LTR
• High gene transduction and high transcription efficiency
• Enables the production of high titer recombinant retroviruses
Amp
r
SD
ψ
pDON-5
Intron + SA
4697 bp
HCMV
IE promoter
MLV R
MCS
HCMV
IE promoter
MLV R
MLV U5
Application
MLV U3
PmaCI (1370)
Apa I (1375)
Sac II (1379)
Not I (1381)
Bgl II (1389)
Cla I (1395)
Bam HI (1401)
Sal I (1407)
Hpa I (1413)
Xho I (1421)
Amp
r
MLV U5
PmaCI (1370)
Apa I (1375)
Sac II (1379)
Not I (1381)
Bgl II (1389)
Cla I (1395)
Bam HI (1401)
Sal I (1407)
Hpa I (1413)
SD
MLV U5
ψ
Intron + SA
pDON-5 Neo
5828 bp
Minimal
SV40 promoter
r
Neo
MLV U5
MLV U3
MLV R
MLV R
• Retrovirus-mediated gene transfer into mammalian cells
3' LTR
3' LTR
Description
The pDON-5 and pDON-5 Neo vectors facilitate both high-efficiency gene transduction
and high expression. They allow higher production of high-titer recombinant retroviruses
than pMEI-5 or pMEI-5-Neo (see Figure 1). By transfecting the vector pDON-5 or pDON-5
Neo vector into an appropriate packaging cell line, the vector expresses transient or
stable transcribed product containing virus packaging signal (psi) and target gene and
selective marker.
ivp/ml
Mammalian Expression Vectors
MCS
5' LTR
Features
8,000,000
3.5
7,000,000
3.0
6,000,000
2.5
5,000,000
2.0
4,000,000
1.5
3,000,000
1.0
2,000,000
0.5
1,000,000
0
0
DON-AI-2 DON-AI-2 MEI-5 Neo
Neo
MEI-5
DON-5 Neo
DON-5
Figure 1: Comparison of Virus Titers (ZsGreen/HT1080)
The ZsGreen gene was inserted into the Bam H I/Hpa I site of the indicated
vectors. G3T-hi cells were transiently transfected and then 2 sets of recombinant
retroviruses were produced for each viral vector.
14
These vectors possess LTR and psi (viral packaging signal), but not the structural genes
necessary for particle formation and replication (gag, pol, and env). These vectors include
a strong cytomegalovirus promoter (HCMV IE) within the U3 region of 5’ LTR and a
human EF1α-derived intron with high splicing activity upstream of the multiple cloning
site. pDON-5 Neo includes the neomycin resistant cassette as a selective marker.
DON-AI-2
Neo
DON-AI-2
MEI-5 Neo
MEI-5
DON-5 Neo
DON-5
Figure 2: ZsGreen Expression Intensity
(relative value to the mean fluorescence intensity of 1 copy/cell)
HT1080 cells were infected with recombinant retroviruses at various dilution rates
using polybrene. Three days after transduction, gene transfer efficiency and ZsGreen
expression intensity were measured using a flow cytometer. Values shown were
normalized using the expression intensity of DON-AI-2 virus as 1.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
Chaperone Plasmid Set
Chaperone Plasmid Set
3340
1 Set
Application
araC
• Promotes correct in vivo folding of expressed recombinant proteins in E. coli
groEL
araB
dnaK
Description
pG-KJE8
11.1 kb
araC
groES
Pzt1
araB
Pzt1
araB
gro ES
pACYC ori
pACYC ori
Cmr
pKJE7
dnaK
groEL
7.2 kb
araC
pG-Tf2
pTf16
8.3 kb
grpE
dnaJ
tetR
tig
gro EL
Cmr
5 kb
tig
pACYC ori
Resistance
Plasmid
pG-KJE8
pGro7
pKJE7
pG-Tf2
pTf16
Chaperone
Promoter Inducer
Marker
dnaK-dnaJ-grpE-groES-groEL araB, Pzt1 L-Arabinose, TetracyclineCmr
groES-groEL
araB
L-Arabinose
Cmr
dnaK-dnaJ-grpE
araB
L-Arabinose
Cmr
groES-groEL-tig
Pzt1
Tetracycline
Cmr
tig
araB
L-Arabinose
Cmr
Plasmids with the Chaperone Plasmid Set work well in combination with the pCold
expression system vectors.
References
Kit Components
2. Nishihara, K., et al. (1998) Appl. Environ. Microbiol. 64(5):1694-1699.
1. Nishihara, K., et al. (2000) Microbiol. 66(3):884-889.
5 plasmids: conc. 10 ng/µL; 100 µL each plasmid
Human gene A (~70 kDa) was expressed in insoluble form when
using pCold I alone. However, the level of soluble expressed protein
increased significantly when the chaperone plasmid pG-Tf2 was
co-expressed with the pCold I construct.
Human gene B (~24 kDa) was not expressed when using pCold I DNA
alone. However, co-expression with the chaperone plasmid pG-Tf2
resulted in expression of high levels of target protein in soluble form.
The combination of Cold Shock Expression Vectors and the Chaperone Plasmid Set often leads to significant improvement in expression level of soluble forms of target proteins.
If sufficient expression or solubilization cannot be achieved using pCold vectors alone, we recommend co-expression with chaperone plasmids. Furthermore, pCold vectorbased expression systems may produce better results by co-expressing chaperone plasmids carrying the tig sequence, such as pG-Tf2 or pTf16, which are included in the
Chaperone Plasmid Set (data not shown).
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
15
Folding
Note that this system cannot be used in combination with chloramphenicol-resistant E.
coli host strains or expression plasmids that carry a chloramphenicol-resistance gene. For
example, E. coli BL21(DE3), which is often used with pET systems, is a compatible host
strain. However, E. coli BL21(DE3) pLysS and BL21(DE3) pLysE, which contain pLysS or
pLysE plasmids that have the pACYC replication origin and the Cmr gene, cannot be used
with this system.
groES
rrnBT1T2
araC
5
araB
pACYC ori
grpE
Cmr
pACYC ori
pGro7
5.4 kb
tetR
dnaJ
The Chaperone Plasmid Set consists of 5 different plasmids, each of which is designed
to express multiple molecular chaperones. Together, they function as a “chaperone
team” to facilitate protein folding. Co-expression of a target protein with one of these
plasmids increases the recovery of soluble proteins. Each plasmid carries an origin of
replication derived from pACYC and a Cmr gene, which allows use with E. coli expression
systems utilizing ColE1-type plasmids with an ampicillin resistance gene as a marker. The
chaperone genes are situated downstream of an araB or Pzt-1 (tet) promoter. Therefore,
expression of target proteins and chaperones can be induced individually if the target
gene is placed under the control of other promoters (e.g. lac). These plasmids also contain
the necessary regulator (araC or tetR) for each promoter.
Cmr
Cmr
Chaperonin GroE
Chaperonin Gro EL
Chaperonin Gro ES
Folding
5
7330
7331
5 mg
0.5 mg
Application
Description
• Facilitates refolding of denatured proteins
Chaperonin GroE is a protein complex composed of GroEL (14 subunits, 57 kDa) and
GroES (7 subunits, 10 kDa). It is thought to support the ability of proteins to form tertiary
structure upon or immediately after translation. GroE is essential to assembly (and
presumably reassembly after denaturation) of protein complexes in vivo. Chaperonin
GroEL and GroES can be used for refolding denatured proteins to recover functional
activity.
Corystein™ (Purothionin) Reagent
Corystein™ (Purothionin) Reagent 7311
5 mg
Application
Description
• Facilitates protein refolding by promoting exchange reactions between disulfide
bonds
Corystein™ (Purothionin) Reagent is a polypeptide purified from wheat endosperm. It
catalyzes the formation of correct disulfide bonds in pro­teins. Corystein™ Reagent can
be used alone or together with thioredoxin on a variety of proteins to re-form disulfide
bonds.
Refolding CA Kit
Refolding CA Kit
Refolding CA Kit
7350 (small)
7351 (large)
Application
25 reactions
1 kit
Kit Components
• Refolding of isolated inclusion body proteins
Description
The Refolding CA Kit uses a novel artificial chaperone technology (licensed from NFRI,
BTRAI, and Ezaki Glico Co, Ltd.) in an easy 2-step procedure for optimizing the refolding
conditions of inclusion body proteins. Optimization allows identification of the best
conditions for correct protein folding and restoration of protein activity.
The Small Kit (Cat. #7350) is supplied with guanidine hydrochloride and DTT for protein
denaturation, four different surfactants that can be added independently to the unfolded
protein solution to protect against molecular aggregation, and highly polymerized
cycloamylose (CA), an artificial chaperone, for surfactant removal and recovery of protein
activity. Overnight incubation of the CA-treated protein is followed by a quick 10-minute
centrifugation. The resulting supernatant contains the refolded protein.
The Large Kit (Cat. #7351) is used for large-scale refolding after initial determination of
with the Small Refolding CA kit, and consists only of denaturant and CA.
7350 (Small Kit)
8 M guanidine hydrochloride (GdmCl)
4 M dithiothreitol (DTT)
4 surfactants:
1% Tween 40
1% Tween 60
1% CTAB (cetyltrimethylammoniumbromide)
1% SB3-14 (myristylsulfobetaine)
200 mM DL-cystine
3% CA (highly polymerized cycloamylose)
2 x 1 mL
50 µL
2 x 1 mL
2 x 1 mL
2 x 1 mL
2 x 1 mL
2 x 0.75 mL
7 x 1.6 mL
7351 (Large Kit)
3% CA (highly polymerized cycloamylose)
8 M guanidine hydrochloride (GdmCl)
6 x 20 mL
2 x 10 mL
References
Related Products
1. Machida, S., et al. (2000) FEBS Lett. 486(2):131-135.
Chaperone Plasmid Set, 3340, p. 15.
2. Sundari, C.S., et al. (1999) FEBS Lett. 443(2):215-219.
3. Daugherty, D.L., et al. (1988) J. Biol. Chem 273(51):33961-33971.
Principle of the Refolding CA Kit
Guanidi
ne
hydrochloride
unfolds inclusion
bodies
16
Surfactants
prevent
protein
aggregation
Highly polymer
ized CA
removes surfactants
and facilitates
otein
pr
refolding
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
Biolo
g icall
y
active protein in
thermodynamically
stable native
conformati
on
High-level Secretion of Recombinant Protein using
the Brevibacillus Expression System
By Michikazu Tanio and Toshiyuki Kohno, Mitsubishi Kagaku Institute of Life Sciences (MITILS), Tokyo, Japan
Analysis of Expression
The obtained colonies were cultured for 1-3 days with 3-5 ml of media in a 14-18 mm
diameter tube (37°C, 200 rpm reciprocal shaking culture). Two kinds of media were
prepared: 2SYNm (2% glucose and 4% soytone and 0.5% yeast extract and 1 mM
calcium chloride and 50 μg/ml neomycin) and TMNm (1% glucose and 1% polypeptone
and 0.5% bonito extract and 0.2% yeast extract and 0.001% iron sulfate and 0.001%
manganese sulfate, 0.0001% zinc sulfate, and 50 μg/ml neomycin). Expression
analysis by SDS-PAGE and western blot with anti-His antibody showed that FD1 could
be detected only in 2SYNm medium and FKBP was produced in both 2SYNm and
TMNm media. 2SYNm medium was selected for large-scale culture due to its ease of
preparation.
The yeast protein expression system using Pichia pastoris is known to be quite efficient
for expression of proteins containing S-S bonds, but expression levels are highly variable
among target proteins.
Large-Scale Culture and Purification
Various types of proteins have been successfully expressed using insect cells or animal
cells, including target proteins that could not be expressed with E. coli systems, but E.
coli remains superior for productivity and cost. However, a major advantage of eukaryotic
expression systems including yeast is the capacity for post-translational modification
such as phosphorylation or glycosylation, although such modifications would be an
obstacle in structural analysis by leading to protein heterogeneity.
Cell-free expression systems can sometimes express cytotoxic proteins, and avoid
metabolic issues when performing selective amino-acid labeling with stable isotopes.
However, cell-free reactions are performed in a reductive environment, thus preventing
expression of proteins containing disulfide bonds. These systems are also of relatively
high cost.
Thus, each heterologous protein expression system has advantages and drawbacks
and selection of an appropriate system must consider both cost and research purpose.
However, there is no satisfactory system suitable for proteins containing disulfide bonds.
In our laboratory, we have analyzed protein structure and function using different
expression systems. The Brevibacillus Expression System was introduced recently. This
expression system, which Takara Bio launched in 2006, has already shown numerous
successes for producing secreted recombinant proteins (1-4). With this system we
could obtain some secreted or cytoplasmic proteins that failed to be expressed with an
E. coli system or a yeast system (5-7). Considering the simple protocol and affordable
cost of the Brevibacillus Expression System, it should be a first choice - before the E. coli
expression system - for recombinant protein expression.
1 ml of bacterial pre-culture incubated in 3-5 ml of 2SYNm medium was added to a 500 ml flask with 100 ml of 2SYNm or SYNm medium (2% glucose and 0.8% soytone and
0.5% yeast extract and 50 µg/ml neomycin) and incubated at 27°C or 37°C at 100 rpm for 1-5 days. The number of culture flasks was increased as needed according to
the desired amount of protein expression and the end use of recombinant target protein.
The expressed proteins were purified by affinity chromatography with TALON® Metal
Affinity Resin (Clontech) after adjusting the culture supernatant pH to 8.
FD1 was subjected to ion exchange and gel filtration chromatography (Fig 1), and FKBP
was purified by gel filtration chromatography only.
Stable Isotopic Labeling
1 ml of the bacterial pre-culture in 2SYNm was added into 500ml flask with 100 ml of
Stable Isotope Labeling C.H.L. Medium (Chlorella Industry Co., Ltd.) added by 50 μg/
ml neomycin and incubated at 27-37°C, 100 rpm for 1-5 days. The labeled proteins
were purified by affinity chromatography and gel filtration chromatography. For amino
acid-selective labeling, the unlabeled C.H.L. medium added by labeled amino acids (100
mg/L) was used as culture media.
In this paper, we describe both recombinant protein production and stable isotope
labeling using the Brevibacillus Expression System.
Methods
Expression and Purification of Two Recombinant Proteins - Human M-Ficolin
Recognition Domain (FD1: Molecular weight 26.8 kDa) and Human FK506
Binding Protein (FKBP: Molecular weight 13 kDa)
Construct Design
After amplification by PCR using Human Universal QUICK-Clone™ cDNA II (Clontech)
as a template, each cDNA encoding the target protein was cloned into the pNCMO2
vector with insertion of a C-terminal 6 x His-Tag. The expression vector constructs were
transformed into Brevibacillus choshinensis Electro-Cells with the electroporation protocol
recommended by the manufacturer's user manual.
Figure 1. Expression and purificaton
of FD1 protein with the Brevibacillus
Expression Sytem.
Lane 1: Culture supernatant after 2 days. Lane
2: FD1 protein purified by Talon. Lane 3: FD1
protein purified by ionic exchange and gel filtration
chromatography.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
17
6
Application Notes
Introduction
A variety of currently used heterologous protein expression systems has been developed,
such as E. coli, yeast, insect cells, animal cells and cell-free systems. Such expression
systems enable the production of proteins that are difficult to isolate from raw material,
and allow introduction of mutations, heavy atoms, and isotopes. The most popular and
first choice in most cases is the E. coli protein expression system, which features high
productivity, ease of use, and relatively low cost. However, many proteins are often
produced in E. coli systems in insoluble inclusion bodies, particularly for proteins with
disulfide(S-S) bonds, secreted proteins, or proteins originating from higher organisms.
Refolding of inclusion bodies or of target proteins secreted to the periplasm can be
performed, but this generally leads to lower production yields of active protein.
Results
Stable Isotope Labeling
Secretion by the Brevibacillus Expression System
Application Notes
6
FD1, which contains two S-S bonds and includes a substrate binding domain of human
M-Ficoline, functions as a foreign substance recognition protein during innate immunity
(Fig 2). Although we have already reported the FD1 crystallographic structure by
secretion of FD1 with the yeast expression system P. pastoris (8-10), at the time of the
original studies we could hardly produce any FD1 derivatives in this yeast expression
system. With the Brevibacillus Expression System, however, we succeeded in the
production of secreted FD1 protein and several FD1 derivatives with normal levels of
activity (6). With regard to expression conditions, we found most efficient production
occurring at 200 rpm for a shaking culture in test tubes and at 100 rpm for rotary
shaking cultures in flasks. We observed lower production at higher rpm levels. The most
appropriate culture temperature was 37°C. However, in one case of a certain FD1 variant,
the protein expressed at 37°C showed no substrate binding activity, even though normal
binding activity occurred
upon culture at 27°C. This
implies that, especially for
the derivatives, irreversible
degeneration might occur
in the stable protein, even
though the target protein
has been secreted efficiently.
Several experiments with
different media compositions
showed that media containing
~0.8 % soytone without
calcium chloride is the most
suitable for production of
secreted proteins. A similar
trend occurred not only for FD1
but also FKBP. On the other
hand, pre-culture from a single
colony with SYNm media
Figure 2. Crystal structure of FD1 protein
under this condition resulted
(PDB ID 2D39) (9).
in notably low growth. Taking
all results into consideration,
we chose our final culture condition for pre-culture as 3-5 ml of 2SYNm medium at 37°C
with shaking (200 rpm) overnight (15-18 h), and for large-scale culture as 100 ml of SYNm
medium at 27°C with rotary shaking (100 rpm) for a few days (2-4 days). Under these
conditions, we could obtain ~10 mg of purified FD1 protein from 1 L culture, slightly
higher than we obtained using the yeast expression system (5-8 mg / L). With activity
analysis of FD1 derivative proteins obtained using the optimized culture conditions
described above for the Brevibacillus Expression System, we have successfully identified
the residues involved in FD1 substrate binding (6).
In other studies, FKBP, a cytoplasmic protein lacking S-S bonds, was secreted with the
Brevibacillus Expression System (5, 7). The culture and purification conditions were
almost the same as described above for FD1 above except that the temperature for largescale culture was 37°C instead of 27°C because of the higher protein stability for FKBP.
The yield of the purified FKBP protein with 2SYNm medium was about 16 mg/L.
In our laboratory, we have successfully used the Brevibacillus Expression System to
produce other cytoplasmic proteins in addition to FKBP. One protein of E. coli origin,
which appeared as a mixture of monomer and dimer due to the S-S bond connecting
one protein molecule to another in the oxidized environment of the medium, could be
obtained as a homogenous population of monomers with this expression system by
adding 10 mM of dithiothreitol (DTT) during the purification steps. In another case with a
protein of human origin, expression occurred in the cytoplasm of E. coli, but the
E. coli-expressed protein was degraded during the extraction process possibly because of
endogenous E. coli protease activity. With the Brevibacillus Expression System, however,
this protein was successfully secreted without degradation. These results suggest that
the Brevibacillus Expression System is useful not only for secretory proteins with S-S
bonds but also for the expression of cytoplasmic proteins.
18
Methods for stable isotope labeling with the Brevibacillus Expression System had not
been established prior to the studies summarized here (5-7). Stable isotope labeling
of a target protein is an indispensable technique for protein structural and functional
analyses, especially for NMR studies. We tried to establish the stable isotope labeling
with Brevibacillus Expression System, motivated to do so by the necessity of NMR analysis
of FD1. We used FKBP as a model protein because the NMR signals for that protein were
already assigned and because FKBP contains all 20 types of amino acids. After performing
protein expression tests with several bacterial minimal media, including M9 minimal
medium, Stable Isotope Probing C.H.L. Medium, manufactured by Chlorella Industry
Co., LTD., Japan, showed the best results in terms of both growth and of expression. The
protein yield of purified FKBP in C.H.L. medium was about 2-13 mg/L culture (at 37°C
for 1-2 days). Considering that 1) about 91% of FKBP amino acids were labeled with 15N
when using 15N Labeling C.H.L. medium, and 2) the 1H-15N HSQC NMR spectrum was
quite similar to the data obtained with E. coli expression systems, we could confirm that
the purified proteins expressed with the Brevibacillus Expression System maintained
their normal folding structure. In addition, we tested special media in which each
individual kind of 15N labeled amino acid was added to the non-labeled C.H.L. medium,
trying a total of 19 kinds of amino acids except proline. As a result of 1H-15N HSQC NMR
spectrum analysis on the labeled protein obtained with these 19 kinds of C.H.L. medium
containing each 15N labeled amino acid, we found that selective labeling is possible with
the Brevibacillus Expression System for nine kinds of amino acid residues (Table 1). On the
other hand, the acidic and aromatic amino acids are metabolized to other amino acids
from the first day of culture. Glycine, isoleucine, leucine, serine or threonine are each
metabolized to other amino acids, with the end products being specific to each residue.
We decided to harvest cysteine-labeled FKBP after 1 day of culture because FKBP was
observed to degrade after 2 days culture. Such degradation was also observed after 3
days culture in the tyrosine-labeled C.H.L medium.
Next, in order to show the result of stable isotopic labeling of FD1, we tried to perform
the experiment in a similar way as described for FKBP labeling. We found that with
the simple C.H.L. medium only, the FD1 yield was not as good, but we obtained better
yield with the special C.H.L. medium (C.H.L.aa) where 100 mg/L of eight kinds of amino
Amino Acids
Metabolism
Metabolism Rate (%)**
Selection
Ala
-
< 20
++
Arg
-
< 35
++
Asn
-
< 30
++
Asp
almost all aa
-
-
Csy
-
< 18
++
Gln
-
< 61
++
Glu
almost all aa
-
-
Gly
Cys, Ser, Trp
-
+
His
-
< 25
++
Ile
Leu, Val
-
+
Leu
Ile, Val
-
+
Lys
-
< 26
++
Met
-
< 16
++
Phe
almost all aa
-
-
Ser
Cys, Gly, Trp
-
+
Thr
Cys, Gly, Ser, Trp
-
+
Trp
almost all aa
-
-
Tyr
almost all aa
-
-
Val
-
< 23
++
Table 1. Amino acid selectivity of Brevibacillus* (7).
Brevibacillus choshinensis HPD31-SP3. **Estimated by comparing average NMR signals obtained
by 1H 15N HSQC NMR spectrum results of 15N labeled FKBP proteins.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
acids (without cysteine), respectively, were utilized out of the nine residues that can be
labeled selectively with the Brevibacillus Expression System. Therefore, for the purpose
of producing selectively-labeled FD1 protein, we decided to use the C.H.L.aa medium
where a target amino acid is labeled with stable isotope, or the C.H.L.aa medium with
the labeled cysteine. This improvement has made the amino acid selective labeling of
FD1 possible, and we have successfully obtained several 1H-15N HSQC NMR spectra on
FD1 (Fig 3A-C). The protein yield of purified FD1 was 2-5.5 mg/L (after 5 days of culture).
Moreover, degradation of cysteine-labeled target protein was not observed in FD1. In
conclusion, we have established a simple and cost-effective Stable Isotope Labeling
method with the Brevibacillus Expression System.
Conclusions
1) Normal S-S bond formation can be expected.
2) Target proteins can be purified directly from the media without need for bacterial lysis.
3) A large amount of target protein can be obtained because they are secreted into the
media rather than sequestered in the bacterial cytoplasm.
4) Some proteins that readily form inclusion bodies when expressed in E. coli are more
likely to maintain their normal folding structure during dilution into the culture
media.
5) Even cytotoxic proteins that exhibit toxicity inside the cells can potentially be
produced efficiently using the Brevibacillus Expression System by being secreted into
the extracellular medium.
6) The Brevibacillus Expression System offers an affordable alternative to eukaryotic
systems. Although eukaryotic expression systems such as insect cells are primarily
used for the secretory production of recombinant protein at present, eukaryotic
systems requires both sophisticated protocols and expensive equipment compared
with the E. coli expression system. From that perspective, the Brevibacillus Expression
System could be recommended as an alternative method for producing proteins
that cannot be easily expressed by E. coli. The Brevibacillus system is compatible
with culture equipment and materials used for E. coli expression. Additionally, NMR
structural analysis of secreted proteins produced with the Brevibacillus Expression
System may become more attractive in the future.
1. Udaka, S., Yamagata, H. (1993) High-level secretion of heterologous proteins by
Bacillus brevis. Methods Enzymol. 217:23-33.
2. Miyauchi, A., Ozawa, M., Mizukami, M., Yashiro, K., Ebisu, S., Tojo, T., Fujii, T., Takagi,
H. (1999) Structural conversion from non-native to native form of recombinant
human epidermal growth factor by Brevibacillus choshinensis. Biosci. Biotechnol
.Biochem. 63(11):1965-1969.
3. Yashiro, K., Lowenthal, J. W., O’Neil, T. E., Ebisu, S., Takagi, H., Moore, R. J. (2001)
High-level production of recombinant chicken interferongamma by Brevibacillus
choshinensis. Protein Expr. Purif. 23(1):113-120.
4. Tanaka, R., Mizukami, M., Ishibashi, M., Tokunaga, H., Tokunaga, M. (2003) Cloning
and expression of the ccdA-associated thiol-disulfide Oxidoreductase (catA) gene
from Brevibacillus choshinensis: stimulation of human epidermal growth factor
production. J. Biotechnol. 103(1):1-10.
5. Tanio, M., Tanaka, T., Kohno, T. (2008) 15N isotope labeling of a protein secreted by
Brevibacillus choshinensis for NMR study. Anal. Biochem. 373(1):164-166.
6. Tanio, M., Kohno, T. (2009) Histidine regulated activity of M-ficolin. Biochem. J.
417(2):485-491.
7. Tanio, M., Tanaka, R., Tanaka, T., Kohno, T. (2009) Amino acid-selective isotope
labeling of proteins for nuclear magnetic resonance study: Proteins secreted by
Brevibacillus choshinensis. Anal. Biochem. 386(2):156-160.
8. Tanio, M., Kondo, S., Sugio, S., Kohno, T. (2006) Overexpression, purification and
preliminary crystallographic analysis of human M-ficolin fibrinogen-like domain. Acta
Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 62(Pt. 7):652-655.
9. Tanio, M., Kondo, S., Sugio, S., Kohno, T. (2007) Trivalent recognition unit of innate
immunity system: crystal structure of trimeric human M-ficolin fibrinogen-like
domain. J. Biol. Chem. 282(6):3889-3895.
10Tanio, M., Kondo, S., Sugio, S., Kohno, T. (2008) Trimeric structure and conformational
equilibrium of M-ficolin fibrinogen-like domain. J. Synchrotron Radiat. 15(Pt. 3):243245.
However, in our experience, some proteins were expressed at low levels and others
could not even be cloned into the vector. Moreover, in order to improve the yield
of the obtained protein, increasing TALON resin volume and re-applying the flowthrough is also required. We believe this implies that some 2SYNm and TMNm medium
components may prevent absorption of His-tags. In the future, we expect that additional
studies on both advantages and drawbacks of Brevibacillus Expression System will lead
to further developments and improvements of this expression system.
Figure 3. 15N stable isotope labeling
with Brevibacillus Expression System.
1H-15N HSQC NMR spectrum results of A) FD1 protein
with 15N-labeled amino acids, B) FD1 protein with
[α-15N] labeled Ala, and C) FD1 protein with [α-15N]
labeled His. It has been known that FD1 protein has 2 S-S
bonds (Fig.2). This experiment was perfomed with a FD1
variant. (F127S/L128S) stays as monomer in solutions,
because wild-type FD1 forms trimers.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
19
6
Application Notes
The production of secreted recombinant protein using the Brevibacillus Expression
System has several advantages:
References
The pCold TF Protein Expression System Produces
Soluble, Active Protein in E. coli
Application Notes
6
The elucidation of protein structure and function continues to be important in the
post-genomic era. An efficient protein production system is critical for obtaining large
amounts of correctly folded recombinant protein for study. E. coli expression systems
are used extensively for production of recombinant proteins, and have two major
advantages over other expression systems: (1) ease of use, and (2) low cost. However,
some recombinant proteins do not fold correctly during expression in E. coli, resulting in
deposits of inactive insoluble protein termed “inclusion bodies”.
Materials and Methods
Series of pCold Vectors
In collaboration with Prof. Masayori Inouye (University of Medicine and Dentistry of
New Jersey), Takara Bio has developed the pCold DNA Vectors, a series of novel protein
expression vectors. The pCold Vectors provide increased in vivo protein yield, purity, and
solubility of expressed recombinant proteins using “cold shock” technology. The cspA
(cold shock protein A) promoter and related elements have been incorporated into these
vectors to up-regulate target protein production at lowered incubation temperatures
(37°C-15°C). This temperature drop also suppresses expression of other cellular proteins,
represses protease activity, and temporarily halts overall cell growth. This process allows
expression of target proteins at high yield, high purity (up to 60% of cellular protein),
and increased solubility as compared with conventional E. coli expression systems.
Co-expression of one or more chaperone proteins during expression of a heterologous
target protein has proven effective for obtaining higher amounts of soluble recombinant
protein (see Takara’s Chaperone Plasmid Set (Cat. # 3340)). This procedure, though, lacks
the convenience of a single transformation step.
pCold TF Vectors
Takara’s pCold TF DNA Vector is a fusion cold shock expression vector that expresses a
molecular chaperone (Trigger Factor (TF)) as a soluble tag. Trigger Factor is a prokaryotic
ribosome-associated chaperone protein (48 kDa) that facilitates co-translational folding
of newly expressed polypeptides. Because of its E. coli origin, TF is highly expressed in
E. coli expression systems. The pCold TF DNA Vector consists of the cspA (cold shock)
promoter plus additional downstream sequences including a 5’ untranslated region (5’
UTR), a translation enhancing element (TEE), a His-Tag sequence, and a multiple cloning
site (MCS). A lac operator is inserted downstream of the cspA promoter to ensure strict
regulation of expression. Additionally, recognition sites for HRV 3C Protease, Thrombin,
and Factor Xa are located between the TF-Tag and the MCS to facilitate tag removal from
the expressed fusion protein. Most E. coli strains can serve as expression hosts.
20
pCold TF DNA Vector combines high-yield cold shock expression technology with Trigger
Factor (chaperone) expression in a single vector to facilitate correct protein folding, thus
enabling efficient soluble protein production for otherwise intractable target proteins.
The following experiment compares results generated using 1) pCold I, 2) pCold I
co-expressed with a Chaperone plasmid, 3) pCold TF, or 4) T7 promoter constructs to
express various proteins.
pCold DNA I and pCold TF DNA cloning and expression procedures* were conducted as
described in this experimental overview:
1) Insert the target gene to the multiple cloning site of the pCold DNA vector for
expression.
2) Transform the E. coli host strain (e.g. BL21) with the expression plasmid and select for
ampr transformants.
3) Inoculate the transformants into medium including 50 µg/mL of ampicillin, and
incubate with shaking at 37°C.
4) At OD600= 0.4 - 0.5, refrigerate the culture at 15°C (without shaking) for 30 minutes.
5) Add IPTG to a final concentration of 0.1-1.0 mM, then continue incubation with
shaking at 15°C for 24 hours.
6) Collect the cells and confirm the expression of the target protein by SDS-PAGE of
soluble and insoluble fractions or by activity assay.
Expression from T7 promoter-driven vectors was performed using a standard protocol
utilizing IPTG induction and subsequent culturing at 37°C.
*Cultivation/induction conditions (culture medium, aeration, timing of induction,
concentration of inducer, cultivation time after induction) should be optimized for each
target protein.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
Results and Discussion
kDa
pCold TF
1 2
pCold +
Chaperone
1 2
pCold
1 2
97
66
1. Cell extract solution
2. Soluble fraction
target protein
co-expressed
trigger factor
*
31
Figure 1: Expression of Soluble Protein A Using the pCold TF
Expression System
Trx
GST
Nus
1 2 3 1 2 3 1 2 3
pCold pCold I pCold +
Chaperone
TF
1 2 3 1 2 3 1 2 3
kDa
45
Figure 1 shows the successful production of enzyme protein A using the pCold TF system.
Upon expression, this protein (estimated molecular weight 29 kDa) was not observed as
a discernable band with either the T7 expression system or even with pCold I (by either
individual expression or chaperone co-expression).
However, the expression of the target protein and target plus tag (29 kDa and 52 kDa)
was observed using pCold TF, and most of the obtained protein was in soluble form.
Subsequent assays confirmed that the expressed enzyme A retains activity even as a
fusion protein (data not shown).
22
97
66
Protein expression using the pCold TF Expression Vector was compared with protein
expression using (1) the pCold DNA I Vector alone, (2) co-expression using the pCold DNA
I Vector with Takara Bio’s Chaperone Plasmid pTf16, and (3) a T7 promoter expression
system which included other solubilization-promoting tags.
1. Cell extract
solution
2. Soluble
fraction
3. Insoluble
fraction
31
22
Figure 2 shows improved expression of soluble protein B using pCold TF. Expression of
soluble enzyme protein B (M.W: ~63 kDa) was not observed using either pCold DNA I
alone or pCold I co-expressed with chaperone proteins, nor with a T7 expression vector
that included other tags for solubilization (Trx Tag [~12 kDa], Nus Tag [~55 kDa], and
GST Tag [~26 kDa]).
However, when the pCold TF DNA Vector was used, the target protein was observed at
an expression level much higher than that achieved with other systems and tags, and
most of the expressed target protein was observed in the soluble fraction. (Note: due to
the presence of various tags, target protein molecular weight appears larger and more
variable than its actual size).
In summary, the pCold TF expression system offers several advantages including
convenience, high yield, and high purity. The pCold TF system is suitable for efficient
soluble protein expression in E. coli of otherwise intractable target proteins.
Figure 2: Increased Expression of Soluble Protein B Using the
pCold Expression System
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
21
6
Applications Notes
*
45
T7
1 2
Unfolding the Potential of Proteins
New refolding technologies are essential for tomorrow’s recombinant proteins
By Joby Marie Chesnick
In vivo, small polypeptides often fold spontaneously into their correct configuration.
However, longer polypeptides have a greater likelihood of folding more slowly and have
greater potential to form partially folded intermediate structures and aggregates that
are non-functional. The timing of polypeptide folding can also influence the success
rate of correct protein folding. For example, outcomes may very if protein folding is
concomitant with ribosomal synthesis or if it is delayed until transport into the cytoplasm
or to cellular organelles.
One of the largest problems encountered by scientists attempting to express
recombinant proteins in bacteria is the formation of inclusion bodies, insoluble
aggregates of misfolded polypeptides that are produced as the bacterium quickly
synthesizes large quantities of the foreign protein. These misfolded protein aggregates
must be unfolded and refolded to assume their correct 3-dimensional structure before
further study or production can proceed. As research efforts have continued to shift
from the investigation of gene structure to the study of protein structure and function,
the importance of studying recombinant proteins has increased. Robust methods for
producing accurately folded structures are therefore essential.
Molecular chaperones
More than 40 years ago, Ferruccio M. Ritossa from the International Laboratory of
Genetics and Biophysics in Naples, Italy, first discovered the heat shock response of
certain proteins through observation of a new puffing pattern in Drosophila buschii
salivary gland polytene chromosomes (1). This puffing was indicative of increased gene
expression of these proteins, which were later termed heat shock proteins (HSPs). HSPs
and related proteins (e.g. DnaK, Hsp40, GrpE; GroEL/S; trigger factor; prefoldin CCT; SecB;
ClpA) are called “molecular chaperones” because they help ensure that polypeptides
assume the correct conformation.
One of the most important functions of chaperones is in aiding the protein-folding
process. Because molecular chaperones coevolved with polypeptides—the presence of
highly conserved sequence data for chaperones and representation of these molecules in
every major taxon, including eukaryotes, eubacteria, archaea, and viruses, suggests that
chaperones are ancient molecules—they provide for a controlled cellular mechanism
by which proteins can reach their most thermodynamically stable 3-dimensional
conformation.
Chaperones accomplish controlled protein folding either by directly binding to conserved
domains in a nascent polypeptide and preventing interactions with other adjacent
protein domains or by providing, through their own 3-dimensional structure, a space
that allows controlled polypeptide folding (2). Folding typically occurs via several
binding and release events with the polypeptide, which are mediated by the hydrolysis
of ATP. But even with all of the chaperone machinery present in a cell, 30% or more of
all synthesized polypeptides cannot fold correctly to form functional proteins (3).
Nonetheless, when there is need to refold proteins to their correct form, molecular
chaperones are essential tools.
22
100
Relative activity, %
Application Notes
6
The development of novel enzymes and proteins relies upon an understanding of their
intrinsic structures. A protein’s 3-dimensional structure dictates the positions of exposed
reactive groups as well as hidden hydrophobic residues, thereby defining its biological
activity. Three-dimensional structure can also influence processes such as protein
trafficking to and between cellular organelles. It is well-documented that several human
diseases including Alzheimer’s Disease, Parkinson’s Disease, and Cystic Fibrosis arise due
to misfolded cellular proteins.
Tween 60
Tween 40
80
60
40
20
0
0
0.2
0.4
0.6
0.8
1.0
Final concentration of CA, %
Figure 1. Comparison of citrate synthase activity following
protein refolding using cycloamylose and Tween 40 or Tween 60.
Full (100%) activity is achieved for refolding of citrate synthase using as little as 0.6%
cycloamylose with Tween 60.
Refolding strategies
The most common strategy currently used to recover active recombinant protein in
vitro from isolated inclusion bodies consists of a three-step process: (1) isolation and
washing of the inclusion bodies, (2) solubilization (i.e., denaturation and unfolding) of
the protein aggregates, and (3) correct refolding of the solubilized protein. The first two
of these steps typically can be performed with high efficiency. However, misfolding and
aggregation of the solubilized protein may complicate the last step.
A number of commercial kits are available for improving the refolding conditions of
inclusion bodies, including Fold-It, developed by Hampton Research; Pro-Matrix Protein
Refolding Kit from Pierce; and Novagen’s Protein Refolding Kit. Their components include
a denaturant to solubilize the inclusion bodies, one or more small-molecule detergents
to maintain the protein in an unfolded configuration and allow refolding through
transient interactions with the protein, and various buffers that differ in parameters that
influence the refolding process, such as pH, redox concentration, and ionic strength. The
degree of successful refolding obtained by the “dilution additive” strategy of these kits
depends on the buffer properties, as well as protein concentration and temperature.
The Refolding CA Kit from Takara Bio offers a novel approach to the dilution additive
method. By using an artificial chaperone—highly polymerized cycloamylose (CA)—
the kit provides correct refolding of aggregated proteins with high effiency. CA aids
refolding through its structure, a single helical V-amylose conformation containing an
anhydrophilic channel-like cavity (4). Inclusion complexes with other molecules such
as detergents can be formed in this space, preventing protein aggregation. In contrast
to the conventional dilution additive method, CA supports stable interactions between
the protein and detergent, and then strips the detergent from the protein–detergent
complex to initiate refolding (5). This scenario results in greater refolding efficiency. CA
can be used in combination with many different types of detergents and is compatible
with redox reagents. In addition, CA can interact with peptides of multiple sizes, is
highly soluble and has a long shelf life in aqueous solution, and accomplishes protein
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
refolding in a short time (i.e., a few hours to overnight). Figure 1 shows the amount
of citrate synthase activity recovered following enzyme refolding using CA. In this
application, full enzyme activity was obtained using 0.6% CA with Tween 60 detergent
(polyoxyethylene sorbitan monostearate) or 1% CA with Tween 40 (polyoxyethylene
sorbitan monopalmitate).
6
The Chaperone Plasmid Set, developed by HSP Research Institute and introduced
into the market by Takara Bio, contains five chaperone-team-containing plasmids for
increasing yields of soluble foreign proteins in vivo. These plasmids carry a pACYC origin
of replication plus a chloramphenicol resistance gene, which allows their use with ColE1type plasmids containing an ampicillin resistance gene. Clones may be selected based on
presence of both a chaperone plasmid and a plasmid containing a target gene of interest.
Figure 2 shows increased amounts of soluble target protein (bacterial protein D), with a
concomitant decrease in insoluble protein, obtained by co-expression of chaperones from
Takara’s pG-KJE8 chaperone plasmid vector.
For obtaining high yields of recombinant proteins, Takara Bio offers plasmids that utilize
the cold-shock protein A (cspA) gene promoter system for gene expression. This system
induces protein expression at low temperatures (15 °C), which suppresses the synthesis
of most other proteins and lowers potentially destructive cellular protease activity. As
a result, up to 60% of all expressed cellular protein is the desired target. Additionally,
high-efficiency metabolic labeling of the protein is possible to facilitate structural
analysis. Large-scale culture and affinity purification (up to 400 mg of protein per liter
of culture) is achievable, making cold shock protein expression suitable for commercial
applications.
Figure 2. Soluble protein production with co-expression of chaperone
genes from Takara’s chaperone plasmid vectors with a bacterial
gene.
A bacterial gene was present at relatively low level in the soluble fraction in absence of
co-expressed chaperones, but solubility was enhanced when chaperones were co-expressed.
Arrow indicates target protein.
The pCold Vector series from Takara Bio allow insertion of a foreign target gene into a
vector for expression using the cspA promoter. The combination of chaperone plasmid
and cold-shock vector technologies provides a system by which high specificity, highyield production of soluble expressed foreign proteins is routinely possible at affordable
costs.
The availability of new and modified proteins and enzymes for medical and biological
research will depend to a large extent on the development of quick, reliable, and costeffective refolding methods.
Reprinted with permission from Modern Drug Discovery, July 2004, 7(7), 67.
Copyright 2004 American Chemical Society.
References:
(1) Ritossa, F. (1962) A new puffing pattern induced by temperature shock and DNP in
Drosophila. Experientia 18:571-573.
(2) Hartl, F.U.; Hayer-Hartl, M. (2002) Molecular chaperones in the cytosol: from nascent
chain to folded protein. Science 295(5561):1852-1858.
(3) Schubert, U., et al. (2000) Rapid degradation of a large fraction of newly synthesized
proteins by proteasomes. Nature 40(6779):770-774.
(4) Machida, S., et al. (2000) Cycloamylose as an efficient artificial chaperone for protein
refolding. FEBS Letters 486(2):131-135.
(5) Daugherty, D.L., et al. (1998) Artificial chaperone-assisted refolding of citrate
synthase. J.Biol. Chem. 273(51):33961-33971.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
23
Application Notes
An alternative in vivo method for obtaining increased soluble expressed protein relies
on chaperone-containing plasmid vectors. In this case, a “team” of chaperone genes,
such as DnaK-DnaJ-GrpE or others, is engineered into a plasmid vector and placed under
control of either an araB or Pzt-1 promoter, with induction by L-arabinose or tetracycline,
respectively. Chaperones are then co-expressed with the target protein of interest in E.
coli. Separate expression of chaperones and target proteins can be accomplished if the
target gene is placed under the control of a different promoter (e.g., lac).
SPP System™ (Single Protein Production System)
1) What strategies can be used to facilitate production of soluble
protein of interest when it is initially expressed in insoluble form?
Possible strategies are:
FAQs
7
• C hange the time at which IPTG is added to induce the expression. It may be necessary
to optimize the most ideal timepoint, which can range from early to late logarithmic
phases of the culture.
• Reduce the concentration of IPTG (down to 0.1 mM).
• Change the strain of host E. coli.
• Extract the cultured cells by sonication in a buffer containing 0.1 to 1% of detergent
(for example, octylglycoside, NP-40, Triton X-100, etc.)
2) How do I select the type of Cold-shock expression vector for SPP
to use?
The Translation Enhancing Element (TEE) facilitates translation when using pCold I
(SP-4), pCold II (SP-4) and pCold III (SP-4). Proteins expressed using pCold I (SP-4) and
pCold II (SP-4) include His-tags and can be affinity-purified with Ni columns. If you do
not desire the presence of any additional amino acid sequences at the N-terminus of the
protein of interest, the pCold I (SP-4) vector is recommended, as it allows cleavage of
the Tag sequence with Factor Xa. Alternatively, pCold IV (SP-4) lacks both TEE and Tag
sequences.
3) What quantity of the protein of interest will be produced from
1 L of culture?
The expression level usually ranges from several mg to several tens of mg per L, although
it varies for each protein of interest. A 3-L culture can typical result in miligrams of
purified protein, provided the protein of interest can be detected by SDS-PAGE followed
by Coomassie brilliant blue (CBB) staining. In the MazF co-expression system, expression
of new endogenous host proteins is suppressed except for target protein. This results in
highly specific production of target protein with target protein comprising up to 90% of
total protein, but may result in growth stress for E.coli itself. Accordingly the expression
yield of a target protein may decrease compared to general expression with pCold DNAs.
24
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
pCold Expression Vectors
1) What are suitable competent cells for pCold vectors for protein
induction and plasmid storage?
Takara’s pCold Vectors utilize an E. coli cold-shock gene promoter. Therefore, most E.coli
strains can be used as a host for protein induction with these vectors.
The following E. coli strains have been tested and are suitable for use with the pCold
Vectors:
2) Where can I obtain sequence information for the pCold Vectors?
Sequencing information for all pCold Vectors has been deposited in GenBank and is
available from the NCBI website (http://www.ncbi.nlm.nih.gov/). GenBank Accession
Numbers for each of the pCold Vectors are listed below:
pCold Vector
pCold I pCold II pCold III pCold IV pCold TF GenBank Accession No.
AB186388
AB186389
AB186390
AB186391
AB213654
3) Can you tell me exactly where the cleavage site for Factor Xa is
located? Is it at Arg-His?
Factor Xa cleaves at the C-terminal side of last amino acid (Arg) of the recognition
sequence Ile-Glu-Gly-Arg. Thus, the Arg-His will be cleaved.
4) How much Factor Xa would be required to cleave 1 mg of
expressed protein?
Takara Bio scientists have used Factor Xa, Restriction Grade (Novagen) for cleavage.
Cleavage conditions vary from protein to protein. For example, Takara Bio scientists
have performed cleavage using 1 U of Factor Xa for 3 µg of a 60 kDa protein during an
overnight incubation at 4°C.
Be sure to optimize the cleavage conditions for each target protein on a small scale prior
to scaling up reactions.
7
To use the translation enhancing element (TEE) provided in pCold III, the target gene
sequence must be in frame with the TEE sequence. The ATG start codon in the TEE is
used for protein expression. Thus, using this vector, your expressed protein will be a
fusion protein. To express only the target protein without TEE, use pCold IV instead.
FAQs
• BL21 strains
• Rosetta (good for eukaryotic genes)
• Origami (good for eukaryotic genes, but grow very slowly)
• JM109
JM109 or DH5α cells are typically used to construct expression plasmids which are
inserted with a target gene. Note, however, that we recommend storing the pCold
Vectors as isolated plasmid DNA, rather than in transformed cells.
5) We intend to use pCold III for expression of our protein. Our
target gene sequence must contain an ATG start codon so that
it can also be used with our PCR primers. However, since the TEE
element provided on pCold III also has an ATG start codon, will
this situation (i.e. the presence of an ATG start site in both TEE
and our target gene sequences) pose a problem for expression?
We recommend using the Nde I restriction site in the MCS for fusion of an insertion
(gene) sequence with a start codon.
For example;
If the sequence of the target gene is this:
ATG AGC GAT AAA ATT ATT CAC.....
Met-Ser-Asp-Lys-Ile-Ile-His-....
and if pCold III containing TEE is used, then fuse the gene into the Nde I site (CATATG) as
shown below, where the gene ATG start site is underlined:
Nde I site
...ATG AAT CAC AAA GTG CAT ATG AGC GAT AAA ATT
ATT CAC.....
The expressed protein will then be:
Met-Asn-His-Lys-Val-His-Met-Ser-Asp-Lys-Ile-IleHis-...
For PCR amplification of the target gene, design the PCR forward primer considering the
points presented above.
6) I recently purchased the pCold I DNA vector and I was
wondering what antibody if any could be used with the
recombinant proteins produced using this vector?
Since most proteins produced using the pCold Vectors will include a His-tag, Takara Bio
recommends using a His-tag antibody. Clontech offers three His-tag antibodies: 6xHis
Monoclonal Antibody (Albumin-Free) (Cat. #631212), 6xHN Polyclonal Antibody (Cat.
#631213), and 6xHis mAb-HRP Conjugate (Cat. #631210). See the Clontech Related
Products section on page 31 for ordering information.
7) Have any of the pCOLD vectors been used for expression in a
cell-free system?
Takara does not have information about use of pCold Vectors for expression in a cell-free
system. However, it is quite possible that general cell-free systems are not suitable for
use with the pCold Vectors since these systems are usually optimized for expression at
37°C.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
25
Refolding CA Kit
FAQs
7
1) What is the difference between the two different sizes of
Refolding CA Kits?
3) Can disulfide bond-reforming components, such as reduced/
oxidized glutathione, be used with Takara’s Refolding CA Kit?
The smaller Refolding CA Kit (Cat. # 7350) has enough reagents for 25 refolding
reactions. This product is sold as an initial kit for refolding smaller amounts of proteins
(typically not more than 0.24 mg protein per reaction; each reaction uses ~24 µl of a 10 mg/ml inclusion body solution) and for testing refolding conditions using
the provided detergents with the CA. The small kit includes denaturant (8 M GdmCl), 4 M DTT, 4 different surfactants (1% Tween 40, 1% Tween 60, 1% CTAB, 1% SB3-14),
100 mM DL-Cystine, and 3% cycloamylose (CA). Note that urea can also be used with our
kit, although it is not included as a component. Please refer to the product manual for
details regarding the kit protocol.
Yes, disulfide bond-forming reagents can be used with the Refolding CA Kit. Note that
our Kit contains DL-Cystine for disulfide bond reformation. The following reference for
use of reduced/ oxidized gluthathione may be helpful:
The large kit (Cat. # 7351) is intended for use after optimal refolding conditions have
been determined using the small kit, and/or when there is need to refold larger amounts
of protein. The large kit includes 10-fold more denaturant (8M GdmCl)(=20 mL) and
cycloamylose (CA)(= 120 mL) than the small kit. The large kit does not include the
detergents, DTT, or DL-Cystine. These reagents are available separately from chemical
reagent suppliers (e.g., Sigma Aldrich).
2) Could you provide the name of a source for DTT, DL-cysteine and
SB3-14 detergent to be used with the Refolding CA Kit? I want to
make sure that we purchase exactly the same items that came in
the small Refolding CA Kit.
Takara uses the following products for this kit:
Component
Supplier
Catalog #
DTT nakalai
14128-91
Sigma
D5545
DL-Cystine nakalai
10316-41
Sigma
C8630
SB3-14 Sigma
T0807
(Note: SB3-14 is registered as N-Tetradecyl-N,N-Dimethyl-3-Ammonio-1Propanesulfonate at Sigma)
Products from the Japanese manufacturer “nakalai” may not be available in your
geographic location. However, Sigma’s products (listed above in parentheses) are
comparable for use with this kit.
26
Machida, S., et al. (2000) Cycloamylose as an efficient artificial chaperone for protein
refolding. FEBS Lett. 486(2):131-135.
This reference recommends using GSH:GSSH at a ratio of 5:1.
4) Are all of the detergents removed from solution after addition
of cycloamylose (CA)?
The surfactants form a precipitant with CA, and the majority of these detergents are
thus removed after the final centrifugation step. However, a small amount of residual
surfactants remain in the final refolding protein solution.
To remove free surfactants, we suggest using BioBeads (Bio-Rad). Add a 1/5 volume of
BioBeads to the final refolding protein solution and stir the mixture for 2 hours. After
stirring, the Bio Beads are removed and the solution is recovered. Using this method,
more than 95% of ionic surfactants and more than 90% nonionic surfactants are
removed. It is, however, difficult to completely remove ALL traces of surfactants in the
refolding protein solution.
5) I do not get a precipitate in the very last step of the protocol
when I use either Tween 40 or Tween 60 with the CA, but I do get
a precipitate if I use either of the other two detergents. Why am I
not getting a precipitate with the Tween 40 or Tween 60?
On rare occasions Tween 40 or Tween 60 will sometimes fail to form a precipitate during
the last step; however, good refolding results are still obtained even though a white
precipitate is not observed. The exact buffer or salt composition and concentration may
influence precipitate formation. However, at present there is not a clear explanation as
to why visible precipitate formation may be variable.
If you do not observe a precipitate, continue with the procedure regardless. You should
perform centrifugation at 15,000 rpm for 10 minutes, and then collect the supernatant
to assess the protein activity.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
Chaperone Plasmid Set
1) We are trying to express a protein which contains a few rare
codons. This means that we need to express the protein in special
strains as Stratagene BL21 Codon plus or Novagen Rosetta. Is
there any incompatibility of your Chaperone Plasmids with these
kinds of strains?
Novagen Rosetta and Stratagene BL21 Codon plus are the only E. coli strains that encode
rare codons. Unfortunately, these strains are chloramphenicol-resistant. Because Takara’s
Chaperone plasmids all contain a pACYC ori and chloramphenicol-resistance gene, these
strains cannot be used as a host for co-expression with the chaperone plasmids.
Takara has successfully folded a 70 kDa protein using a combination of pCold Vectors
(which utilize cold shock expression technology for high yield-high purity protein
expression) in conjunction with one of our Chaperone Plasmids.
3) After I have used the Chaperone Plasmids for correct folding of
my target protein in vivo, can I successfully purify the expressed
target protein without co-purification of the chaperone protein?
Normally I use a His-tag to purify my expressed proteins, but a Histag can interact with dnaK.
Takara often uses His-tags to purify target proteins folded in vivo using the Chaperone
Plasmids, and has never experienced chaperone protein contamination, such as with
dnaK, with target proteins that are purified using His-tag/His-bind resin.
For target protein purification using His-bind resin, be sure to use an appropriate binding
buffer for purification with the proper concentration of NaCl and imidazole.
However, since chaperone proteins such as DnaK or DnaJ do tend to non-specifically bind
to the resin, it may be somewhat difficult to purify GST-fusion proteins by affinity column
purification. In this case, we recommend using buffer that contains ATP and Mg2+, or
using an ATP-agarose resin for release of protein from the column. For further details of
these procedures, please refer to the following references:
MYu, S., et al. (1992) Involvement of the chaperonin dnaK in the rapid degradation of
a mutant protein in Escherichia coli. EMBO J. 11(1):71-77.
Zylicz, M., et al. (1984) Purification and properties of the Escherichia coli dnaK
replication protein. J. Biol. Chem. 259(14):8820-8825.
Takara uses mainly BL21 or BL21 (DE3) as host strains with these plasmids.
Although Takara does not have specific recommendations for E.coli strains, some
generalizations may be made regarding the optimal combination of target expression
vector and chaperone team used. For example, the chaperone teams of groES-groEL and/
or dnaK-dnaJ-grpE may be more useful than tig (Trigger Factor) to fold proteins which
are expressed using pET vectors, regardless of the kind of target protein.
However, in general, we recommending testing all five chaperone plasmids for each
target protein in order to optimize results.
6) Is DNA sequence information available for the Chaperone
Plasmids?
Takara’s Chaperone Plasmids are currently available under a license agreement between
TAKARA BIO INC. and HSP Research Institute, Inc. As a result, to obtain the sequences of any of these plasmids, customers are required to complete and submit a Sequence
Request Form. This form is available on the Chaperone Plasmid Set product page at
www.clontech.com/takara. Click on the orange box in the ordering area at the bottom
of the Chaperone Plasmid Set product page to obtain the form. Upon approval of the
request, the sequence information will be forwarded to you.
7) Are all chaperone genes encoded on an individual Chaperone
Plasmid expressed in the same ratio?
Chaperone genes which are under the control of the same promoter in an individual
Chaperone Plasmid will be expressed in the same ratio. For example, DnaK, DnaJ and
GrpE, which are encoded on the plasmid pKJE7, will all be expressed in the same ratio in
cells. The following references provide SDS-PAGE data for chaperone co-expresssion:
Nishihara, K., et al. (1998) Chaperone coexpression plasmids: differential and
synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an
allergen of Japanese cedar pollen, Cryj2, in Escherichia coli. Appl. Environ. Microbiol.
64(5):1694-1699.
Nishihara, K., et al. (2000) Overexpression of trigger factor prevents aggregation of
recombinant proteins in Escherichia coli. Appl. Environ. Microbiol. 66(3):884-889.
8) Is it possible to use the Chaperones Plasmids to aid in vitro
translation?
Takara’s Chaperone Plasmid Set is designed to aid only in the folding of expressed
proteins in vivo. We do not have any information as to whether the Chaperone Plasmid
Set can be used to aid in vitro translation.
4) Are restriction maps available for any of the vectors in the
chaperone plasmid set?
Restriction maps for Takara’s Chaperone Plasmids are not available.
However, information is available for those restriction enzymes which cut each plasmid
only once. These enzymes are listed below. The number in ( ) is the 5’ terminal of each
digestion site.
• -pKJE7; Kpn I (5660), Nhe I (6606), Sac I (4157), Sca I (410), Spe I (5632)
• -pG-KJE8; Nhe I (10608), Sac I (4157), Sca I (410), Xho I (8181)
• -pGro7; Bgl II (3929), EcoR I (5462), Hind III (3941), Nhe I (4880), Sca I (410),
Sma I(3832), Xba I (4040)
• -pG-Tf2; BamH I (1979), Hind III (8208), Nhe I (7268), Sal I (4493), Sca I (6271), Sma I
(2786), Spe I (4511), Xho I (1)
• -pTf16; BamH I (3323), Bgl II (1535), Hind III (1523), Kpn I (1529), Nhe I (583), Sca I
(4601), Sma I (2534), Xba I (1424)
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
27
7
FAQs
2) We have a 64 kDa protein that we have expressed in E. coli.
After induction there is a reasonable amount of soluble protein,
but most of this protein is not functional, probably because is
not properly folded. Can Takara’s Chaperone Plasmids help us to
overcome problems associated with expressing a larger protein
such as this one?
5) Has Takara tested any strains of E. coli with these plasmids? Do
they have a recommendation for strains that work best with the
plasmids?
Takara’s Protein Sequencing and Analysis Products
Takara offers a wide variety of Protein Fragmentation products as well as N-terminal deblocking and sequence
determination and C-terminal sequence determination products. The fragmentation products are used for analysis of the
primary structure of proteins and peptides.
Product Name
N-terminal and C-terminal analysis
Pfu Aminopeptidase I
Pfu Pyroglutamate
Aminopeptidase
Pfu Methionine
Aminopeptidase
Fragmentation of Proteins
Pfu N-acetyl Deblocking
Aminopeptidase
(Ac-DAP)
Protease Inhibitor
Protein Sequencing and Analysis Products
8
28
Application
•Liberates N-terminal amino acids up to
X-Pro from proteins and peptides
•Removal of pyroglutamic acids from the
N-terminal of proteins and peptides
•Deblocking of N-terminal pyroglutamates
of proteins and peptides for sequence
analysis using Edman degradation
•Liberates the N-terminal methionine
residues from proteins and peptides
•N-Terminal deblocking
•N-terminal sequence analysis of blocked
proteins or peptides
Description
Pfu Aminopeptidase I is a thermostable exo-type aminopeptidase,
isolated from Pyrococcus furiosus and produced as a recombinant
protein, which liberates the N-terminal amino acid from proteins and
peptides.
Pfu Pyroglutamate Aminopeptidase liberates the N-terminal
pyroglutamic acid from proteins and peptides. This enzyme may work
well with some intact, non-denatured proteins and the denaturation
step may be unnecessary in these instances.
Pfu Methionine Aminopeptidase specifically liberates only the
N-terminal methionine residue from Met-X-Y when X is Ala, Gly, Ser,
Thr, Pro or Val. This enzyme does not liberate the N-terminal Met
when the N-terminus sequence is Met-Met-Y or Met-Met-Met-Y. It is
not active toward formyl-methionine.
Pfu N-acetyl Deblocking Aminopeptidase (Ac-DAP) is a unique exotype aminopeptidase that first liberates blocking groups, such as
formyl, acetyl, and myristyl, and then releases the first and subsequent
amino acids from proteins and peptides until it reaches the first X-Pro
bond.
Arginylendopeptidase
•Fragmentation of proteins and peptides
required from primary structure analysis
Arginylendopeptidase cleaves peptide bonds at the carboxyl side of
arginine residues found in pro­teins and pep­tides. Arginylendopeptidase
is also known as mouse submaxillary pro­tease D or as mouse EGF
binding protein C.
Asparaginylendopeptidase
•Fragmentation of proteins and peptides
required for primary structure analysis
Asparaginylendopeptidase specifically cleaves peptide bonds on the
carboxyl side of asparagine residues found in proteins and peptides.
Glycosylated asparagine residues are not cleaved.
Endoproteinase Asp-N
•Fragmentation of proteins and peptides
required for primary structure analysis
Endoproteinase Asp-N is a metalloprotease that hydrolyzes peptide
bonds on the amino side of Asp and Cys oxidized to cysteic acid. If
cysteine is reduced or alkylated, the enzyme will cleave only the amino
side of Asp residues.
Pfu Protease S
•Fragmentation of proteins and peptides
required for primary structure analysis
Pfu Protease S is an endo-type serine protease with broad recognition of
native and denatured proteins. Cleavage occurs mainly on the carboxy
side of peptide bonds of hydrophobic amino acid residues.
•Calpain protease inhibitor
Calpastatin is an endogenous protease in­hib­i­tor that acts specifically
on calpain calcium-dependent cysteine pro­tease. It consists of four
repetitive sequences of 120 to 140 amino acid residues (domains I, II,
and IV), and an N-ter­mi­nal non-homologous sequence (L). The prod­uct
consists of highly purified recombinant human calpastatin domain I .
Calpastatin
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
PrimeSTAR® GXL DNA Polymerase
PrimeSTAR® GXL DNA Polymerase PrimeSTAR® GXL DNA Polymerase R050A R050B 250 Units 1,000 Units
• Successful, Robust, High-Yield PCR regardless of Conditions
• Long PCR: Amplify products up to 30 kb (human genomic DNA), 40 kb
(lambda DNA), or 13.5 kb (human cDNA)
• Use on Standard and Challenging Templates alike: Outstanding
performance on GC-rich or AT-rich templates and targets containing
repeats
• Can be used with Samples Containing Excess Nucleic Acid: Tolerates a
wide range of template quantity, including high levels of template
that inhibit other high-fidelity DNA polymerases
• Next Generation Sequencing (NGS) studies involving deep sequencing
of the same region in many samples
PrimeSTAR® GXL
Company I Company T
M 1 2 3 4 M M 1 2 3 4 M 1 2 3 4 M Features
• Highest Processivity: among commercially available high-fidelity DNA
polymerases
• Antibody-Mediated Hot-Start Formulation
• Proven Performance: as reported in peer-reviewed literature
Comparison of Amplification of GC-rich targets using PrimeSTAR GXL and other commercially available high-fidelity DNA polymerases.
Excellent results were achieved using PrimeSTAR® GXL DNA Polymerase without requiring special
buffers or reaction conditions. Description
Capable of outstanding performance for both routine high-fidelity PCR and challenging
templates or reaction conditions, PrimeSTAR GXL DNA Polymerase is the most robust
high-fidelity PCR enzyme commercially available. It provides high yield, high specificity,
and high accuracy for not only standard reactions, but also excels in PCR with GC-rich
templates, in the presence of eXcess template, and for amplification of Long products up
Template: Human genomic DNA (100 ng / 50 μl
Template: T. thermophilus HB8 genomic DNA
reaction) 1. APOE gene 746 bp (GC content 74%) 2. TGF (10 ng / 50 μl reaction) 3. 2029 bp (GC content
74%) 4. 4988 bp (GC content 74%)
β 1 gene 2005 bp (GC content 69%)
PrimeSTAR® Max DNA Polymerase
PrimeSTAR® MAX DNA Polymerase PrimeSTAR® MAX DNA Polymerase R045A R045B Applications
•
•
•
•
Use Whenever Accuracy and Fidelity is Critical
Cloning and Expression Studies
Structure-Function Studies
Analyses that involve Evolutionary Inferences (SNP analyses, evolutionary development experiments, etc.)
• Whenever Fast PCR Cycles are Needed, such as High-Throughput
Studies
Features
•
•
•
•
•
Highest Fidelity: of any commercially available PCR polymerase
Fastest Extension Speed: means less time required for PCR cycles
Convenient Premix: assemble reactions in less time
Antibody-Mediated Hot-Start Formulation
Proven Performance: as reported in peer-reviewed literature
100 Rxns 400 Rxns PCR. Since it is formulated as a premix that supports hot-start PCR, it's also excellent
for high-throughput experiments. When you need fast reaction times and/or highly
accurate amplification for cloning and expression, structural studies, or evolutionary
analyses, PrimeSTAR Max DNA Polymerase is the enzyme of choice.
PrimeSTAR Max DNA Polymerase is suitable for reactions occurring in the presence
of excess nucleic acid. Such extraneous DNA in a reaction mix ordinarily inhibits PCR
amplification when using conventional polymerases because the amount of effective
polymerase available is limited by nonspecific binding. The superior processivity
of PrimeSTAR Max DNA Polymerase prevents such inhibition by excess nucleic acid,
resulting in a much higher success rate for PCR with minimal optimization of conditions
required. Furthermore, the antibody-mediated hot start formulation prevents false
initiation events during the reaction assembly due to mispriming and primer digestion.
Since PrimeSTAR Max DNA Polymerase is configured as a 2-fold premix containing
reaction buffer and dNTP mixture, it allows rapid preparation of reactions and is useful
for high-throughput applications.
PrimeSTAR® Max
Company T
Company S
M 1 2 3 4 5 6 7 8 M M 1 2 3 4 5 6 7 8 M 1 2 3 4 5 6 7 8
Description
PrimeSTAR Max DNA Polymerase is a unique high-performance DNA polymerase for
PCR. PrimeSTAR Max DNA Polymerase has the highest fidelity and fastest extension
speed of any commercially available enzyme, along with extremely high sensitivity,
processivity, and specificity. It includes an elongation factor to provide efficient priming
and extension, greatly reducing the time required for annealing and extension steps. As
a result, PrimeSTAR Max DNA Polymerase can be used for exceptionally fast high-speed
Good amplification was observed for
products up to 6 kb using an extension time
of 10 sec. with PrimeSTAR Max.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
Template: λ DNA (1 ng/50 μl reaction)
Target size: 0.5 kb (lane 1) , 1 kb (lane 2), 2 kb (lane 3), 4 kb (lane
4), 6 kb (lane 5), 8 kb (lane 6), 10 kb (lane 7), 12 kb (lane 8)
Extension time: 10 sec
29
9
High Fidelity PCR Reagents
to 30 kb (GXL). Simplify your PCR and save time by relying on one enzyme system works
regardless of conditions, with minimal optimization required.
PrimeSTAR GXL DNA Polymerase includes a modified PrimeSTAR HS enzyme and an
additional elongation factor which in combination provide unsurpassed processivity.
PrimeSTAR GXL DNA Polymerase has outstanding performance in reactions containing
excess nucleic acid. Such extraneous DNA in a reaction mix ordinarily inhibits
PCR amplification by conventional polymerases because the amount of effective
polymerase available is limited by nonspecific binding. The superior processivity
of PrimeSTAR GXL DNA Polymerase prevents such inhibition by excess nucleic
acid, resulting in a much higher success rate for PCR with minimal optimization of
conditions required. Furthermore, the antibody-mediated hot start formulation
prevents false initiation events during the reaction assembly due to mispriming and
primer digestion.
Applications
Takara Related Products
Ordering Information
Takara Related Products
10
Product Name
Product No.
Quantity
Ligation
T4 DNA Ligase
2011A
12,500 U (100 Weiss units)
DNA Ligation Kit, Mighty Mix
6023
1 kit (75-150 rxns)
DNA Ligation Kit, Version 2.1
6022
50 Rxns
Cloning Vectors
pBR322 DNA
3050
25 µg
pUC18 DNA
3218
25 µg
pUC19 DNA
3219
25 µg
pUC118 DNA
3318
25 µg
pUC119 DNA
3319
25 µg
Protein Sequencing and Analysis
Pfu Pyroglutamate Aminopeptidase
7334
10 mU
Pfu Methionine Aminopeptidase
7335
20 mU
Pfu Aminopeptidase
7336
0.5 mg
Pfu N-acetyl Deblocking Aminopeptidase
7340
50 µg
Arginylendopeptidase
7308
0.5 mg
Asparaginylendopeptidase
7319
0.2 mU
Endoproteinase Asp-N
7329
2 µg
Pfu Protease S
7339
500 U
Electrophoresis
Mupid®-exU Electrophoresis System
AD140
1 Unit
Mupid®-2plus Electrophoresis System
AD110
1 Unit
Mupid® One Electrophoresis System
AD160
1 Unit
Power Supply for Mupid®-2Plus
AD111
1 Unit
Retroviral Transduction
RetroNectin® Recombinant Human Fibronectin Fragment
T100A
0.5 mg
RetroNectin® Recombinant Human Fibronectin Fragment
T100B
2.5 mg
RetroNectin® Precoated Dish
T110A
10 Dishes
Ladders
1 kb DNA Ladder (Dye Plus)
3426A
100 Rxns
100 kb DNA Ladder (Dye Plus)
3422A
100 Rxns
20 bp DNA Ladder (Dye Plus)
3420A
100 Rxns
200 bp DNA Ladder (Dye Plus)
3423A
100 Rxns
250 bp DNA Ladder (Dye Plus)
3424A
100 Rxns
500 bp DNA Ladder (Dye Plus)
3425A
100 Rxns
High Fidelity PCR Enzymes
PrimeSTAR® GXL DNA Polymerase
R050A
250 Units
PrimeSTAR® GXL DNA Polymerase
R050B
1000 Units
PrimeSTAR® Max DNA Polymerase
R045A
100 Rxns
PrimeSTAR® Max DNA Polymerase
R045B
400 Rxns
Protease Inhibition
Calpastatin
7316
3 mg
Takara has been manufacturing high quality restriction enzymes for over 30 years
and offers more than 90 restriction enzymes to meet your cloning needs.
Visit our web site at
www.clontech.com/takara
to view these and other products.
30
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
Clontech Related Products
Ordering Information
Product Name
Product No.
Quantity
TALON Prepacked
HisTALON Gravity Columns
635655
5 columns
HisTALON Gravity Columns Purification Kit
635654
20 purifications
TALON Single Step Columns (20 ml)
635632
10 columns
HisTALON Superflow Cartridges (5 x 1 ml)
635650
5 cartridges
HisTALON Superflow Cartridge (1 x 5 ml)
635683
1 cartridge
HisTALON Superflow Cartridges (5 x 5 ml)
635682
5 cartridges
HisTALON Superflow Cartridge Purification Kit (5 x 1 ml)
635649
20 purifications
HisTALON Superflow Cartridge Purification Kit (5 x 5 ml)
635681
5 purifications
His60 Ni Superflow Resin
His60 Ni Superflow
635659
10 ml
635660
25 ml
635661
4 x 25 ml
635662
250 ml
635663
2 x 250 ml
635664
4 x 250 ml
His60 Ni Prepacked
His60 Ni Gravity Columns
635657
5 columns
His60 Ni Gravity Columns Purification Kit
635658
20 purifications
His60 Ni Superflow Cartridges (5 x 1 ml)
635675
5 cartridges
His60 Ni Superflow Cartridge (1 x 5 ml)
635680
1 cartridge
His60 Ni Superflow Cartridges (5 x 5 ml)
635679
5 cartridges
His60 Ni Superflow Cartridge Purification Kit (5 x 1 ml)
635674
20 purifications
His60 Ni Superflow Cartridge Purification Kit (5 x 5 ml)
635678
5 purifications
In-Fusion® HD Cloning System Liquid Kits
In-Fusion® HD Cloning System
639645
10 Rxns
639646
50 Rxns
639647
100 Rxns
639692
96 Rxns
In-Fusion® HD Cloning System CE
639636
10 Rxns
639637
50 Rxns
639638
100 Rxns
639693
96 Rxns
Antibodies
c-Myc Monoclonal Antibody 631206 200 μg HA-Tag Polyclonal Antibody 631207 100 μg c-Myc Monoclonal Antibody-Agarose Beads 631208 1 ml 6xHis mAb-HRP Conjugate 631210 100 μl 6xHis Monoclonal Antibody (Albumin-Free) 631212 200 μg 6xHN Polyclonal Antibody 631213 200 μl In-Fusion® HD Cloning System EcoDry™ Kits
In-Fusion® HD EcoDry™ Cloning System
639684
8 Rxns
639685
24 Rxns
639686
48 Rxns
639688
96 Rxns
Protease Inhibitor
ProteoGuard EDTA-Free Protease Inhibitor Cocktail 635672
100 μL
635673
10 x 100 μL
Clontech Related Products
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
11
31
Takara Product Offering
Take advantage of Takara’s broad product portfolio and expert manufacturing capabilities to
increase reliability and reproducibility, and to reduce your costs. Takara also offers custom,
bulk and OEM services.
Protein Research
Molecular Biology
• Restriction and modifying enzymes
• Cloning vectors
• DNA/RNA ladders and MW markers
• Electrophoresis salts and buffers
PCR Polymerases and Reagents
• Polymerases
• Real-Time (qPCR) reagents
• Reverse transcriptases
• Primers and buffers
• Protease Inhibition
• Protein Folding & Expression
• Protein Sequencing and Analysis
Glycobiology
• Glycobiology Enzymes and Reagents
• Glycobiology Kits
Cellular Biology
• Antibodies
• EIA kits
Notice to Purchaser. Your use of these products and technologies is subject to compliance with any applicable licensing requirements described on the
product’s web page at http://www.clontech.com/takara. It is your responsibility to review, understand and adhere to any restrictions imposed by such
statements. Unless otherwise specified, other trade names are also the trademarks or registered trademarks of various companies.
TAKARA BIO INC.
TB 633353 Dist
www.clontech.com/takara