pCold GST载体说明

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

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 everse 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--‐

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。

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--‐transferases, 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 rotease 的识别序列，可从融合蛋白质中去除标签。HRV 3C P rotease 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 c annot b e e fficiently e xpressed i n E. c oli b ecause 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 Jersey), Takara Bio has developed a system for improving protein expression in E. coli t hat i s b ased 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--‐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 a nd s olubility o f t he f used 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 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 e xpression.

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℃), 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） Insert 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 nserted i n--‐frame w ith t he G ST t ag s equence. （2） Transform the host E. coli cells with the plasmids, and select 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.

（6） Confirm the presence, 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 increase 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 sed 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 rotease (Cat. #7360).

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(+)

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

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