Cold Expression – TAKARA

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pCold TF DNA Vector is a fusion cold shock expression vector that expresses Trigger Factor (TF) chaperone as a soluble tag
(48 kDa) which facilitates co-translational folding of newly expressed polypeptides. The vector has a His-Tag sequence, and a multicloning 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 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

Elucidation of protein structure and function maintains an important role in post-genomic sequencingand analysis studies. An efficient protein production system is critical for obtaining large amounts ofcorrectly 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 ofexpression systems: (1) ease of use, and (2) low cost. However, some recombinant proteins do notfold 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 expressionvectors. The pCold Vectors provide increased in vivo protein yield, purity, and solubility for ex-pressed recombinant proteins using “cold shock” technology. More specifically, the cspA (coldshock protein A) promoter and related elements have been incorporated into these vectors to up-regulate target protein production at lowered incubation temperatures (37oC-15oC). This temperaturedrop 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 cellularprotein), and increased solubility as compared with conventional E .coli expression systems. Co-expression of one or more chaperone proteins during expression of a heterologous targetprotein has proven effective for obtaining increased amounts of soluble recombinant protein inE. coli (see Takara’s Chaperone Plasmid Set [Cat. # 3340]). This procedure, though, lacks theconvenience of a single transformation step.Takara’s pCold TF DNA Vector is a fusion cold shock expression vector that expresses TriggerFactor (TF) chaperone as a soluble tag. Trigger Factor is a prokaryotic ribosome-associatedchaperone 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 pColdTF 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 amulticloning site (MCS). A lac operator is inserted downstream of the cspA promoter to ensure strictregulation of expression. Additionally, recognition sites for HRV 3C Protease, Thrombin, and FactorXa are located between TF-Tag and the Multiple Cloning Site (MCS) and function to facilitate tagremoval from the expressed fusion protein. Most E. coli strains can serve as expression hosts. ThepCold TF DNA Vector provides cold shock technology for high yield protein expression combinedwith Trigger Factor (chaperone) expression to facilitate correct protein folding, thus enabling efficientsoluble protein production for otherwise intractable target proteins.

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