pCold TF DNA
Overview
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. 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 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 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. E. coli strains can serve as expression hosts.
- Highly efficient protein expression using cold shock technology
- High yield of active protein due to the trigger factor chaperone solubility-promoting fusion tag
pCold TF DNA Vector Map
pCold TF DNA Vector Map.
Improved solubility of recombinant proteins with the pCold TF DNA Vector.
Expression resulting in improved solubility of recombinant proteins. Takara Bio's pCold TF DNA Vector is a cold shock expression vector that expresses the trigger factor (TF) chaperone as a fusion tag for improved solubility of recombinant proteins. The TF tag facilitates co-translational folding of newly expressed polypeptides and is expressed at high levels in E. coli expression systems. In this experiment, enzyme protein B (~63 kDa) was expressed in E. coli using the pCold TF DNA Vector and a variety of other expression systems. Poor yields of the protein in a soluble form (green circles) were obtained when it was expressed with the pCold DNA I Vector (either alone or coexpressed with a chaperone protein) or with a T7 expression vector that included either the Trx (~12 kDa), Nus (~55kDa) or GST (~26 kDa) solubilization tag (see figure). In contrast, a large yield of soluble enzyme protein B (red circle) was obtained when it was fused to the TF tag [~48 kDa] and expressed using the pCold TF DNA Vector. The level of protein B in the soluble fraction was much higher when expressed as a TF fusion protein than when expressed as a fusion with other tags. (Note: The molecular weights of the fusion proteins were larger than the size of the native target protein.)
Gerlined, S. et al. EMBO J. 14:4939–4948 (1995).
Qing, G. et al. Cold-shock induced high-yield protein production in Escherichia coli. Nature Biotechnol. 22:877–2004 (2004).
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pCold TF DNA Vector Map
pCold TF DNA Vector Map.
Improved solubility of recombinant proteins with the pCold TF DNA Vector.
Expression resulting in improved solubility of recombinant proteins. Takara Bio's pCold TF DNA Vector is a cold shock expression vector that expresses the trigger factor (TF) chaperone as a fusion tag for improved solubility of recombinant proteins. The TF tag facilitates co-translational folding of newly expressed polypeptides and is expressed at high levels in E. coli expression systems. In this experiment, enzyme protein B (~63 kDa) was expressed in E. coli using the pCold TF DNA Vector and a variety of other expression systems. Poor yields of the protein in a soluble form (green circles) were obtained when it was expressed with the pCold DNA I Vector (either alone or coexpressed with a chaperone protein) or with a T7 expression vector that included either the Trx (~12 kDa), Nus (~55kDa) or GST (~26 kDa) solubilization tag (see figure). In contrast, a large yield of soluble enzyme protein B (red circle) was obtained when it was fused to the TF tag [~48 kDa] and expressed using the pCold TF DNA Vector. The level of protein B in the soluble fraction was much higher when expressed as a TF fusion protein than when expressed as a fusion with other tags. (Note: The molecular weights of the fusion proteins were larger than the size of the native target protein.)
