|5' 测序引物及序列:||pCold Forward: 5′-ACGCCATATCGCCGAAAGG-3′|
|3' 测序引物及序列:||pCold Reverse 5′-GGCAGGGATCTTAGATTCTG-3′|
|载体标签:||GST Tag（N-端）, HRV 3C蛋白酶切位点（N-端）,His Tag（N-端）|
pCold GST DNA在冷休克载体的基础上，整合了来源于Schistosoma japonicum的谷胱甘肽S-转移酶(glutathione S-transferases, GST)可溶性标签。通过GST在目的蛋白质N末端的融合表达，可提高融合蛋白质的稳定性和可溶性。 本制品在cspA启动子的下游插入了5’非编码区（5’-UTR）、翻译增强元件（TEE）、His标签、GST标签和多克隆位点（MCS）（下图）。此外，在cspA启动子下游还插入了可以严格调控目的基因表达的lac operator。 GST标签融合蛋白质可进行高亲和性纯化。在GST标签和多克隆位点（MCS）之间插入了高特异性HRV 3C Protease 的识别序列，可从融合蛋白质中去除标签。HRV 3C Protease的最适温度为4～5℃，可以在温和的条件下进行目的蛋白质的标签去除反应。 pCold 系列载体的启动子是大肠杆菌来源的，所以大部分大肠杆菌都可以作为表达宿主使用。 Effective protein expression systems are essential for analyzing protein structure and function. Expression systems using E. coli as a host are widely used for recombinant protein production. Although E. coli expression systems are easy to work with, some genes cannot be efficiently expressed in E. coli because of protein insolubility and toxicity. In collaboration with Professor Masayori Inouye (University of Medicine and Dentistry of New Jersey), Takara Bio has developed a system for improving protein expression in E. coli that is based on cold shock technology (pCold). With this system, the culture is shifted to a low incubation temperature, thereby halting bacterial growth and the expression of most E. coli -derived proteins. Simultaneously, the expression of cold shock proteins is specifically induced. With the pCold vector, target gene expression is driven by the promoter of cspA , an E. coli cold shock gene. Thus, the pCold expression system can significantly improve protein expression, purity, and solubility1. The expression vector pCold GST DNA was developed by incorporating glutathione S-transferase (GST) derived from Schistosoma japonicum as a soluble tag2, 3. The proteins expressed using this vector have an N-terminal GST tag, which can improve the stability and solubility of the fused protein. The pCold GST vector includes a 5' untranslated region (5' UTR), translation enhancing element (TEE), his tag, GST tag, and multiple cloning site (MCS) downstream of the cspA promoter (Figure 1). In addition, a lac operator has been inserted downstream of the promoter to allow precise control of gene expression. Finally, because the pCold vector series uses an E. coli promoter, virtually any strain of E. coli can be used as a host for protein expression. High affinity purification of the GST-tagged fusion proteins expressed using pCold GST is possible. Furthermore, a highly specific HRV 3C protease recognition sequence has been inserted between the GST tag and MCS, allowing removal of the GST tag from the recombinant protein. Because the optimum reaction temperature for HRV 3C protease is low (4 - 5℃), tag cleavage can be performed under moderate conditions. Protocol Cloning and expression of a target gene: （1） Insert the target gene fragment into the multiple cloning site of pCold GST vector. Be sure that the sequence of the fragment is inserted in-frame with the GST tag sequence. （2） Transform the host E. coli cells with the plasmids, and select transformants on an agar plate containing ampicillin. （3） Inoculate LB medium containing 50 - 100 μg/ml of ampicillin with Amp+ transformant clones, and incubate with shaking at 37℃. （4） When the OD600 of the culture reaches 0.4 - 0.8, quickly cool the culture to 15℃ in ice water, and let stand for 30 minutes. （5） Add IPTG to a final concentration of 1 mM, and incubate with shaking at 15℃ for 12 - 18 hours. （6） Confirm the presence, amount, and solubility of the target protein using SDSPAGE or activity measurement. Notes: 1. By optimizing the host strain, culture, and expression induction conditions (e.g., culture medium and temperature, degree of aeration and agitation, timing of induction, IPTG concentration, culture conditions after induction, etc.), it may be possible to increase the expression level and solubility of the target protein. 2. GST affinity purification resins such as Clontech's Glutathione-Superflow Resin (Cat.#635607/635608) can be used to purify GST-tagged fusion proteins. 3. The GST tag can be cleaved using HRV 3C protease (Cat. #7360).