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保存条件 :-20℃
保质期 :6个月
英文名 :pCold-GST
库存 :大量
供应商 :钰博生物
规格 :1ug/5ug
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.ProtocolCloning 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-2016COMMENT http://www.biofeng.com/COMMENT This file is created by Vector NTICOMMENT ORIGDB|GenBankCOMMENT 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\PromoterBASE 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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