| 出品公司: | TAKARA |
|---|---|
| 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 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。 |
| 产品目录号: | #3372 |
| 稳定性: | 稳表达 |
| 组成型/诱导型: | 诱导型（IPTG) |
| 病毒/非病毒: | 非病毒 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
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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