pCold-GST
编号 载体名称
北京华越洋生物VECT4560 pCold-‐GST
pCold G ST载体基本信息
载体名称: pCold-‐GST
质粒类型: 大肠杆菌表达载体
高拷贝/低拷贝: 低拷贝
巳时五行属什么
克隆方法: 限制性内切酶；多克隆位点
启动子: cspA
载体大小: 5097 b p
5' 测序引物及序列: pCold F orward: 5′-‐ACGCCATATCGCCGAAAGG-‐3′
3' 测序引物及序列: pCold R ever 5′-‐GGCAGGGATCTTAGATTCTG-‐3′
载体标签: GST T ag（N-‐端）, H RV 3C蛋白酶切位点（N-‐端）,His T ag（N-‐
梁冀端）
载体抗性: 氨苄青霉素（Ampicillin）
克隆菌株: DH5alpha等
表达菌株: 任意E.coli菌株
备注: pCold-‐GST载体表达GST融合蛋白，GST标签可以提高目的
蛋白的可溶性，提高目的蛋白的纯度。图片识图
稳定性: 稳表达
组成型/诱导型: 诱导型（IPTG)
病毒/非病毒: 非病毒
pCold G ST载体质粒图谱和多克隆位点信息
pCold G ST载体简介
pCold G ST D NA在冷休克载体的基础上，整合了来源于Schistosoma j aponicum的谷胱甘肽S-‐转移酶(glutathione S-‐transferas, G ST)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 o perator。
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 P rotea 的识别序列，可从融合蛋白质中去除标签。HRV 3C P rotea 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 esntial for analyzing protein structure and function. Expression systems using E. coli as a host are widely ud for recombinant protein production. Although E. coli expression systems are easy to work with, some genes c annot b e e fficiently e xpresd i n E. c oli b ecau o f p rotein i nsolubility a nd t oxicity. In c ollaboration w ith P rofessor M asayori I nouye (University o f M edicine a nd D entistry o f New Jery), Takara Bio has developed a system for improving protein expression in E. coli t hat i s b ad o n c old s hock t echnology (pCold). W ith t his s ystem, t he c ulture
i s s hifted 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 i mprove p rotein e xpression, p urity, a nd s olubility1.
The expression vector pCold GST DNA was developed by incorporating glutathione S-‐transfera (GST) derived from Schistosoma japonicum as a soluble tag2, 3. The proteins expresd using this vector have an N-‐terminal GST tag, which can improve the stability a nd s olubility o f t he f ud p rotein.
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 inrted downstream of the promoter to allow preci control of gene ex
荞麦怎么吃pression. Finally, becau the pCold vector ries us an E. coli promoter, virtually any strain of E. coli can be ud as a host for protein e xpression.
High affinity purification of the GST-‐tagged fusion proteins expresd using pCold GST is possible. Furthermore, a highly specific HRV 3C protea recognition quence has been inrted between the GST tag and MCS, allowing removal of the GST tag from the recombinant protein. Becau the optimum reaction temperature for HRV 3C protea is low (4 -‐ 5℃), t ag c leavage c an b e p erformed u nder m oderate c onditions.
Protocol
Cloning a nd e xpression o f a t arget g ene:
（1） Inrt the target gene fragment into the multiple cloning site of pCold GST vector. Be s ure t hat t he s equence o f t he f ragment i s i nrted i n-‐frame w ith t he G ST t ag s equence. （2） Transform the host E. coli cells with the plasmids, and lect transformants on an agar p late c ontaining a mpicillin.
（3） Inoculate LB medium containing 50 -‐ 100 μg/ml of ampicillin with Amp+ transformant c lones, a nd i ncubate w ith s haking a t 37℃.
（4） When t he O D600 o f t he c ulture r eaches 0.4 -‐ 0.8, q uickly c ool t he c ulture t o 15℃ in ice w ater, a nd l et s tand f or 30 m inutes.
（5） Add IPTG to a final concentration of 1 mM, and incubate with shaking at 15℃ for 12 -‐ 18 h ours.
作文仙人掌（6） Confirm the prence, amount, and solubility of the target protein using SDSPAGE or a ctivity m easurement.
Notes:
1. B y o ptimizing t he h ost s train, c ulture, a nd e xpression i nduction c onditions (e.g., c ulture medium and temperature, degree of aeration and agitation, timing of induction, IPTG concentration, culture conditions after induction, etc.), it may be possible to increa the expression l evel a nd s olubility o f t he t arget p rotein.
2. GST affinity purification resins such as Clontech's Glutathione-‐Superflow Resin (Cat.#635607/635608) c an b e u d t o p urify G ST-‐tagged f usion p roteins.
3. T he G ST t ag c an b e c leaved u sing H RV 3C p rotea (Cat. #7360).
报答歌词其他大肠杆菌表达载体：
pBV221 ptdTomato pET-52b(+) pAmCyan pDsRed-Express2 pBV220 pCold-GST pColdS-SUMO pCold TF pCold IV pCold III pCold II
pCold I pE-SUMO pCold-ProS2 pBAD102/D-TOPO pBAD202/D-TOPO pACYC184 pBAD/Thio-TOPO pBad/Myc-His C pBad/Myc-His B pBad/Myc-His A pBad/His C pBad/His B pBad/His A pBAD-TOPO pET-23b(+) pET-23a(+)
pET-23c(+) pET-23(+) pET-12b(+) pET-12c(+)
pET-12a(+) pET-11b(+) pET-11a(+) pET-11c(+) pBad24 pQE-82L pQE-81L pQE-80L
pQE-32 pQE-9 pQE-16 pQE-31
pQE-60 pQE-70 pQE-40 pET-51b(+)
pET-50b(+) pET-49b(+) pET-48b(+) pET-47b(+)
读论语有感pET-26b(+) pET-32a(+) pET-21b(+) pET-22b(+)
pET-14b pET-16b pET-15b pET-19b
pET-20b(+) pET-21d(+) pET-21c(+) pET-21b(+)
pET-21a(+) pET-24a(+) pET-24d(+) pET-25b(+)
pET-27b(+) pET-28a(+) pET-30a(+) pET-42a(+)
pET-43.1c(+) pET-43.1b(+) pET-43.1a(+) pET-44a(+)
pET-44c(+) pET-46 EK/LIC pET-37b(+) pTrcHis2 C pTrcHis2 B pTrcHis2 A pET303/CT-His pET302/NT-His pRSET-CFP pRSET-EmGFP pRSET-BFP pGFPuv
pET300/NT-DEST pET301/CT-DEST pGEM-T pBad43
pGEX-4T-3 pGEX-5X-2 pBlueScript SK(+) pG-Tf2
pG-KJE8 pGro7 pET-SUMO pSE380
pET-17b pET102/D-TOPO pCDFDuet-1 pMAL-p5x
pTf16 pET-28c(+) pBluescript II SK(+) pET-30b(+) pSUMO pProEX HTc pProEX HTb pProEX HTa pKD3 pKD13 pKD46 pTYB1
梦到打死蛇pTYB2 pTWIN2 pBluescript II KS(-) pTYB12
pMAL-p5e pACYCDuet-1 pEGM-11ZF(+) pEGM-7ZF(+) PinPoint Xa-3 PinPoint Xa-2 PinPoint Xa-1 pSP73
pSP64 pTWIN1 pTYB11 pTXB1
pET-5b(+) pBad/gIII C pBad/gIII B pBad/gIII A pET-5a(+) pMal-p4X pMal-p2G pkk223-3
pkk232-8 pCYB1 pEZZ18 pBAD18
pMAL-c5x pMal-p2E pMal-p2X pET-44 EK/LIC pET-43.1 EK/LIC pET-41 EK/LIC pMal-c4X pTrcHis B
pET-31b(+) pET-3b(+) pET-41a(+) pGEX-3X
pGEX-4T-2 pETDuet-1 pGEX-4T-1 pTrc99a
pET-28b(+) pET-His pALEX a,b,c pACYC177
pBR322 pKD4 pKD20 pMXB10
pEcoli-6xHN-GFPuv pKJE7 pRSET B pGEX-KG
pGEX-2T pRSFDuet-1 pCOLADuet-1 pTrcHis C pTrcHis A pET-41b(+) pET-42b(+) pET-3a(+) pGEX-6P-3 pGEX-6P-2 pGEX-6P-1 pGEX-5X-3 pGEX-5X-1 pGEX-2TK pRSET A pMal-c2G
pMal-c2E pMal-c2X pRSET C pQE-30
pET-45b(+) pET-44b(+) pET-42c(+) pET-41c(+) pET-40b(+) pET-33b(+) pET-39b(+) pET-32 EK/LIC pET-32 Xa/LIC pET-32c(+) pET-32b(+) pET-30 Xa/LIC pET-30 EK/LIC pET-30c(+) pET-29c(+) pET-29b(+) pET-29a(+) pET-24c(+) pET-24b(+) pET-24(+)
pET-23d(+) pET-11d(+) pBad33
