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Table of contents
I. Description (2)
II. Components (2)
III. Vector map (3)
IV. Storage (3)
接地
电缆V. Protocols (3)
VI. Multiple cloning site (4)
VII. Application (4)
VIII. Q&A (5)
IX. Appendix (6)
X. Related Products (10)
XI. References (11)
Elucidation of protein structure and function maintains an important role in post-genomic sequencing I. Description
自动化测试
脚本and analysis studies. An efficient protein production system is critical for obtaining large amounts of
correctly folded recombinant protein for study. E. coli expression systems, which are used exten-
sively for the production of recombinant proteins, offer two major advantages over other types of
expression systems: (1) ease of use, and (2) low cost. However, some recombinant proteins do not
fold correctly during expression in E. coli, and result in deposits of inactive insoluble protein termed
"inclusion bodies".
In collaboration with Prof. Masayori Inouye (University of Medicine and Dentistry of New Jersey,
USA), 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 for ex-
pressed recombinant proteins using "cold shock" technology. More specifically, the csp A (cold马胶配方的大全
shock protein A) promoter and related elements have been incorporated into these vectors to up-
regulate target protein production at lowered incubation temperatures (37o C-15o C). This temperature
drop also suppresses expression of other cellular proteins 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 increased amounts of soluble recombinant protein in
E. coli (see Takara's Chaperone Plasmid Set [Cat. # 3340]). This procedure, though, lacks the
convenience of a single transformation step.
Takara's pCold TF DNA Vector is a fusion cold shock expression vector that expresses Trigger
Factor (TF) chaperone as a soluble tag. Trigger Factor is a prokaryotic ribosome-associated
chaperone protein (48 kDa) which facilitates co-translational folding of newly expressed polypep-
tides. Because of its E. coli origin, TF is highly expressed in E. coli expression systems. The pCold
TF DNA Vector consists of the csp A promoter plus additional downstream sequences including a 5'
untranslated region (5' UTR), a translation enhancing element (TEE), a His-Tag sequence, and a
multicloning site (MCS). A lac operator is inserted downstream of the csp A promoter to ensure strict
regulation of expression. Additionally, recognition sites for HRV 3C Protease, Thrombin, and Factor
Xa are located between TF-Tag and the Multiple Cloning Site (MCS) and function to facilitate tag
removal from the expressed fusion protein. Most E. coli strains can serve as expression hosts. The
pCold TF DNA Vector provides cold shock technology for high yield protein expression combined
with Trigger Factor (chaperone) expression to facilitate correct protein folding, thus enabling efficient
soluble protein production for otherwise intractable target proteins.
II. Components
pCold TF DNA Vector 25 µg
< Available E. coli host strains >
Most E. coli strains can be used as expression hosts for Takara's pCold DNA Vector series since
these vectors utilize the E. coli csp A (cold shock protein gene) promoter.
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Fig.1 pCold TF D N A : Vector Map
数据
监测III. Vector map:1M
3G I pCold TF DNA
5,769 bp
bp C o l E 1o r i A p m A ’UTR lac csp a l c I csp A 3’UTR
Factor Xa site Thrombin site HRV 3C Protease site Trigger Factor (TF)His s Tag TEE IV. Storage:-20°C (for shipping and storage)
V. Protocol:
How to express the target gene;
The cultivation / induction conditions (culture medium, culture temperature, aeration, timing of
induction, concentration of an inducer, cultivation time after induction) should be examined for each
target protein.
The example of general method is shown below.
1) Insert the target gene to the multicloning site of pCold DNA to construct the plasmid for
expression.
2) Transform li host strain (e.g. BL21) with the plasmid of expression, and select the
transformants on the selection plate including ampicillin.
3) Inoculate the transformant in the medium including 50 µg/ml of ampicillin, and culture at 37°C with
shaking.
4) At OD 600= 0.4 - 0.5, refrigerate the culture solution at 15°C and leave to stand for 30 minutes.
5) Add IPTG at the final concentration of 0.1- 1.0 mM, and continue the culture with shaking at 15°C
for 24 hours.
6) Collect the cells, and confirm the expression of target protein with SDS-PAGE in soluble and
煅后焦insoluble fractions or activity assay.
By selection of li host strains for expression and optimization of cultivation / induction condi-
tions (culture medium, culture temperature, aeration, timing of induction, concentration of an inducer,
cultivation time after induction), the expression level and the degree of soluble expression are
improvable. The tag sequence at the N-termini can be cut and removed by Factor Xa, Thrombin, and
HRV 3C Protease. GeneBank Accession No. AB213654
VI. Multiple cloning site:
pCold TF DNA (Code. 3365)VII. Application
Protein expression using pCold TF DNA was compared against expression using (1) the pCold
DNA I Vector alone, (2) co-expression using the pCold DNA I Vector with Takara's Chaperone
Plasmid pTf16, and (3) a T7 promoter expression system which included experiments using other
tags for solubilization. pCold DNA I and pCold TF DNA Vectors were transformed separately into
their protocols. Expression from T7 promoter-driven vectors was additionally conducted using a
general procedure involving addition of IPTG and subsequent culturing at 37o C.
(1) Example 1: Successful protein expression resulting in soluble form
The expression of enzyme protein A (estimated molecular weight 29 kDa) was not verified as an exact band around at the estimated molecular weight, 29 kDa, with the expression system utilizing T7 promoter or even with pCold I DNA (either individual expression or co-expression with chaper- one). On the other hand, the expression of target protein (29 kDa and 52 kDa) was verified in case of using pCold TF DNA, and most of the obtained protein was in soluble form. It was confirmed that the expressed enzyme protein A has the enzyme activity even in the form of a fusion protein.
Fig.2 Expression of enzyme protein A
pCold TF pCold I pCold+ T7 chaperone
kDa 1 2 1 2 1 2 1 2
97-
66-45-
31-22-
*1: cell extract solution
2: solulde fraction @ : target protein * :Co-expressed trigger factor
@
@
@@
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2) Example 2: Expression resulting in improved levels of soluble protein.
Expression of soluble enzyme protein B (M.W: ~63 kDa) was not able to be obtained using either
pCold DNA I alone or 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]). Alternatively, when pCold TF DNA Vector was used, most of the expressed target protein
was verifiable as a soluble fraction and present at an expression level much higher than with other
tags. (Note: The molecular weight of the target protein was observed as larger than its real size due
to fused expression with each tag.)
1: Cell extract solution
2: Soluble fraction
3: Insoluble fraction
Fig.3 Expression of enzyme protein B
VIII. Q&A Q1: What parameters should be examined when the expressed protein is insoluble?
A1: The optimal conditions for cultivation and induction vary depending on a kind of expressed protein. The cultivation and induction conditions should be determined by referring to the
following points:
- Change the induction timing. It should be examined within early and late logarithmic growth
phase.
tf2o
- Change the concentration of inducer (IPTG) within 0.1-1 mM.
- Examine the cultivation time after induction (Generally 15°C, 24 hours is the most
appropriate.)
- Change the conditions for aeration.
Q2: What remedial procedures should be taken in case that no protein is expressed or the expres-sion level is low?
A2: It is recommended to examine again the conditions for cultivation and induction (Refer to the
above Q1.), or change the li strain.
Q3: What host strains have been confirmed to work with pCold Vectors?
A3: BL21, Rosetta TM, Origami TM from Novagen, Inc. BL21 is most commonly used as host.
Origami TM lacks the trx/gor gene and allows the formation of disulfide bond in cytoplasm at high level. Accordingly, the solubility and refolding of expressed protein are facilitated.
Rosetta TM contains a plasmid which supplies tRNAs corresponding to the codons that are rarely used li. It enables the universal transcription of the genes which are restricted by the
codon usage li.
Q4: Can E. Coli retaining a pCold TF vector that contains target gene be stored at 4°C on a plate?
A4: We don't recommend the 4°C storage on a plate because it causes a possible leak of target protein in the cell. Pick the colony from the plate promptly, prepare a glycerol stock and store at
-80°C.
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