pCold-GST

pCold-GST

载体基本信息

出品公司:	Ybscience
	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 bp
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）
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-GST载体质粒图谱和多克隆位点信息

 

pCold-GST载体简介

 

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).

 

载体序列

LOCUS       pCold\GST               5097 bp    DNA     circular     27-NOV-2016
COMMENT     http://www.biofeng.com/
COMMENT     This file is created by Vector NTI
COMMENT     ORIGDB|GenBank
COMMENT     VNTDATE|-11521228|
COMMENT     VNTDBDATE|-11521228|
COMMENT     LSOWNER|
COMMENT     VNTNAME|pCold GST|
COMMENT     VNTAUTHORNAME|Demo User|
FEATURES             Location/Qualifiers
     terminator      complement(3993..4012)
                     /vntifkey="43"
                     /label=Transcription\terminator
     primer          4005..4024
                     /vntifkey="27"
                     /label=pCold-R
     primer          complement(4872..4890)
                     /vntifkey="27"
                     /label=pCold-F
     misc_feature    complement(4798..4809)
                     /vntifkey="21"
                     /label=Factor\Xa\Site
     misc_feature    complement(4810..4827)
                     /vntifkey="21"
                     /label=His\Tag
     misc_feature    complement(4828..4842)
                     /vntifkey="21"
                     /label=TEE
     misc_feature    complement(4852..4855)
                     /vntifkey="21"
                     /label=SD
     misc_feature    complement(4111..4134)
                     /vntifkey="21"
                     /label=HRV\3C\Protease
     CDS             complement(4141..4791)
                     /vntifkey="4"
                     /label=GST
     CDS             164..1246
                     /gene="lacI"
                     /codon_start=1
                     /transl_table=11
                     /product="lactose operon repressor"
                     /protein_id="BAD35135.1"
                     /db_xref="GI:51090249"
                     /vntifkey="4"
                     /label=lacI
     gene            164..1246
                     /gene="lacI"
                     /vntifkey="60"
     rep_origin      complement(1403..2017)
                     /vntifkey="33"
                     /label=ColE1\ori
                     /note="ColE1 origin"
     CDS             complement(2177..3037)
                     /gene="Amp"
                     /codon_start=1
                     /transl_table=11
                     /product="beta-lactamase"
                     /protein_id="BAD35134.1"
                     /db_xref="GI:51090248"
                     /vntifkey="4"
                     /label=Amp
     gene            complement(2177..3037)
                     /gene="Amp"
                     /vntifkey="60"
     misc_feature    3224..3697
                     /vntifkey="21"
                     /label=M13\IG
                     /note="M13 intergenic region"
     3'UTR           complement(3896..4040)
                     /gene="cspA"
                     /vntifkey="50"
                     /label=cspA
                     /note="cspA 3'UTR"
     misc_feature    complement(4048..4107)
                     /gene="cspA"
                     /vntifkey="21"
                     /label=cspA
                     /note="MCS = Multiple cloning sites contain the follow restriction sites: NdeI, SacI, KpnI, XhoI, BamHI, EcoRI, HindIII, SalI, PstI, XbaI"
     promoter        complement(5017..5083)
                     /gene="cspA"
                     /vntifkey="30"
                     /label=cspA\Promoter
BASE COUNT     1277 a      1298 c      1206 g      1316 t 
ORIGIN
        1 gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg 
       61 cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat 
      121 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt 
      181 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca 
      241 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa 
      301 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt 
      361 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg 
      421 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc 
      481 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta 
      541 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt 
      601 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg 
      661 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc 
      721 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac 
      781 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt 
      841 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa 
      901 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc 
      961 ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt 
     1021 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca 
     1081 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag 
     1141 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 
     1201 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 
     1261 atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca 
     1321 acccagtcag ctccttccgg tgggcgcggg gcatgactat gtgagcaaaa ggccagcaaa 
     1381 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 
     1441 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 
     1501 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 
     1561 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 
     1621 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 
     1681 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 
     1741 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 
     1801 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 
     1861 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 
     1921 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 
     1981 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 
     2041 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 
     2101 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 
     2161 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 
     2221 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 
     2281 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 
     2341 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 
     2401 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 
     2461 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 
     2521 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 
     2581 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 
     2641 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 
     2701 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 
     2761 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 
     2821 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 
     2881 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 
     2941 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 
     3001 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 
     3061 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 
     3121 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gcctgatgcg 
     3181 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag 
     3241 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 
     3301 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 
     3361 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 
     3421 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg 
     3481 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 
     3541 ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta 
     3601 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 
     3661 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc 
     3721 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc 
     3781 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 
     3841 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctgcgc 
     3901 attctcattg cacccaaatt tattcttcac aaaaataata atagatttta ttacgcgatc 
     3961 gattatttat ttcctgaaaa caaataaaaa aatccccgcc aaatggcagg gatcttagat 
     4021 tctgtgcttt taagcagaga ttacctatct agactgcagg tcgacaagct tgaattcgga 
     4081 tccctcgagg gtaccgagct ccatatgtcc cgggccctgg aacagaactt ccagatccga 
     4141 ttttggagga tggtcgccac caccaaacgt ggcttgccag ccctgcaaag gccatgctat 
     4201 atacttgctg gatttcaagt acttatcaat ttgtgggata gcttcaatac gttttttaaa 
     4261 acaaactaat tttgggaacg catccaggca cattgggtcc atgtataaaa caacatcaag 
     4321 agcgtcatac aacatgaagt caggatgggt tacatgatca ccatttaaat atgttttatg 
     4381 acataaacga tcttcgaaca ttttcagcat ttcaggtagc ttgctaagaa aatcaacttt 
     4441 gagagtttca aagtctttac tatatgcaat tctcgaaaca ccgtatctaa tatccaaaac 
     4501 cgctccttca agcattgaaa tctctgcacg ctcttttgga caaccaccca acatgttgtg 
     4561 cttgtcagct atataacgta tgatggccat agactgtgtt aatttaacat caccatcaat 
     4621 ataataagga agattgggaa actccaaacc caattcaaac tttttgtttc gccatttatc 
     4681 accttcatcg cgctcataca aatgctcttc atatttttct tcaagatatt ccaaaagaag 
     4741 tcgagtgggt tgcacaaggc ccttaatttt ccaataacct agtatagggg acatgtgcct 
     4801 accttcgata tgatgatgat gatgatgcac tttgtgattc atggtgtatt acctcttaat 
     4861 aattaagtgt gcctttcggc gatatggcgt gctttacaga ttttgaagcg ttaaaggaat 
     4921 gtgcactacg aggggtatca acgataactc ttgaagggac ttgccttact acactggata 
     4981 tgcgctagca catcaaattg ttatccgctc acaatttgat gtgcattaag ccacgcattg 
     5041 gcgggtgatg caacaattat ttttcatatt tatgattaat cggccacacc attcctt 
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