pMal-p2X大肠杆菌表达载体说明

pMal-p2X

编号 载体名称

北京华越洋生物VECT4910 pMal-­‐p2X

pMalp2X载体基本信息

载体名称: pMal-­‐p2X, p Malp2X, p Mal p2X

质粒类型: 大肠杆菌蛋白表达载体

表达水平: 高

启动子: Tac

克隆方法: 多克隆位点，限制性内切酶

载体大小: 6721 b p

5' 测序引物及序列: MalE引物: 5'-­‐GGTCGTCAGACTGTCGATGAAGCC-­‐3';

MBP-­‐F: 5'-­‐gatgaagccctgaaagacgcgcag-­‐3'

3' 测序引物及序列: pBad-­‐R: 5'-­‐gatttaatctgtatcagg-­‐3';

M13-­‐F: 5'-­‐TGTAAAACGACGGCCAGT-­‐3'

载体标签: N-­‐MBP, N-­‐Factor X a

载体抗性: Ampicillin 氨苄

备注: 融合表达麦芽糖结合蛋白MBP，蛋白定位于细胞周质。 稳定性: 瞬时表达 Transient

组成型: 诱导表达

病毒/非病毒: 非病毒

pMalp2X载体质粒图谱和多克隆位点信息

pMalp2X载体简介

pMAL™系统是一种高效的蛋白融合表达及纯化系统。pMAL载体含有编码麦芽糖结合蛋白（Maltose B inding P rotein, M BP）的大肠杆菌malE基因，其下游的多克隆位点便于目的基因插入，表达N端带有MBP的融合蛋白。通过"tac"强启动子和malE翻译起始信号使克隆基因获得高效表达，并进一步利用MBP对麦芽糖的亲和性达到用Amylose柱对融合蛋白的一步亲和纯化。

The s ystem u ses t he p MAL v ectors w hich a re d esigned s o t hat i nsertion i nterrupts a l acZα gene a llowing a b lue-­‐to-­‐white s creen f or i nserts o n X-­‐gal (5). p MAL-­‐c2 s eries h as a n e xact deletion of the malE signal sequence, resulting in cytoplasmic expression of the fusion protein. pMAL-­‐p2 series contains the normal malE signal sequence, which directs the fusion protein through the cytoplasmic membrane. pMAL-­‐p2 fusion proteins capable of being exported can be purified from the periplasm. Between the malE sequence and the polylinker there is a spacer sequence coding for 10 asparagine residues. This spacer insulates M BP f rom t he p rotein o f i nterest, i ncreasing t he c hances t hat a p articular f usion will bind tightly to the amylose resin. The vectors also include a sequence coding for the recognition site of a specific protease. This allows the protein of interest to be cleaved from MBP after purification, without adding any vector-­‐derived residues to the protein (6). For this purpose, the polylinker includes a restriction site superimposed on the sequence coding for the site of the specific protease. This is where the gene of interest is

inserted. A n E coR I s ite i n t he s ame r eading f rame a s t hat o f λgt11 a nd a n umber o f o ther useful sites are present directly downstream. The vectors also include the M13 origin of DNA replication which allows the production of single-­‐stranded DNA for sequencing and mutagenesis b y i nfecting w ith M13KO7 h elper p hage.

pMalp2X载体序列

ORIGIN

1 CCGACACCAT CGAATGGTGC AAAACCTTTC GCGGTATGGC ATGATAGCGC CCGGAAGAGA 61 GTCAATTCAG GGTGGTGAAT GTGAAACCAG TAACGTTATA CGATGTCGCA GAGTATGCCG 121 GTGTCTCTTA TCAGACCGTT TCCCGCGTGG TGAACCAGGC CAGCCACGTT TCTGCGAAAA 181 CGCGGGAAAA AGTGGAAGCG GCGATGGCGG AGCTGAATTA CATTCCCAAC CGCGTGGCAC 241 AACAACTGGC GGGCAAACAG TCGTTGCTGA TTGGCGTTGC CACCTCCAGT CTGGCCCTGC 301 ACGCGCCGTC GCAAATTGTC GCGGCGATTA AATCTCGCGC CGATCAACTG GGTGCCAGCG 361 TGGTGGTGTC GATGGTAGAA CGAAGCGGCG TCGAAGCCTG TAAAGCGGCG GTGCACAATC 421 TTCTCGCGCA ACGCGTCAGT GGGCTGATCA TTAACTATCC GCTGGATGAC CAGGATGCCA 481 TTGCTGTGGA AGCTGCCTGC ACTAATGTTC CGGCGTTATT TCTTGATGTC TCTGACCAGA 541 CACCCATCAA CAGTATTATT TTCTCCCATG AAGACGGTAC GCGACTGGGC GTGGAGCATC 601 TGGTCGCATT GGGTCACCAG CAAATCGCGC TGTTAGCGGG CCCATTAAGT TCTGTCTCGG 661 CGCGTCTGCG TCTGGCTGGC TGGCATAAAT ATCTCACTCG CAATCAAATT CAGCCGATAG 721 CGGAACGGGA AGGCGACTGG AGTGCCATGT CCGGTTTTCA ACAAACCATG CAAATGCTGA 781 ATGAGGGCAT CGTTCCCACT GCGATGCTGG TTGCCAACGA TCAGATGGCG CTGGGCGCAA 841 TGCGCGCCAT TACCGAGTCC GGGCTGCGCG TTGGTGCGGA TATCTCGGTA GTGGGATACG 901 ACGATACCGA AGACAGCTCA TGTTATATCC CGCCGTTAAC CACCATCAAA CAGGATTTTC 961 GCCTGCTGGG GCAAACCAGC GTGGACCGCT TGCTGCAACT CTCTCAGGGC CAGGCGGTGA 1021 AGGGCAATCA GCTGTTGCCC GTCTCACTGG TGAAAAGAAA AACCACCCTG GCGCCCAATA 1081 CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA CGACAGGTTT 1141 CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TAAGTTAGCT CACTCATTAG 1201 GCACAATTCT CATGTTTGAC AGCTTATCAT CGACTGCACG GTGCACCAAT GCTTCTGGCG 1261 TCAGGCAGCC ATCGGAAGCT GTGGTATGGC TGTGCAGGTC GTAAATCACT GCATAATTCG 1321 TGTCGCTCAA GGCGCACTCC CGTTCTGGAT AATGTTTTTT GCGCCGACAT CATAACGGTT 1381 CTGGCAAATA TTCTGAAATG AGCTGTTGAC AATTAATCAT CGGCTCGTAT AATGTGTGGA 1441 ATTGTGAGCG GATAACAATT TCACACAGGA AACAGCCAGT CCGTTTAGGT GTTTTCACGA 1501 GCACTTCACC AACAAGGACC ATAGCATATG AAAATAAAAA CAGGTGCACG CATCCTCGCA 1561 TTATCCGCAT TAACGACGAT GATGTTTTCC GCCTCGGCTC TCGCCAAAAT CGAAGAAGGT 1621 AAACTGGTAA TCTGGATTAA CGGCGATAAA GGCTATAACG GTCTCGCTGA AGTCGGTAAG 1681 AAATTCGAGA AAGATACCGG AATTAAAGTC ACCGTTGAGC ATCCGGATAA ACTGGAAGAG 1741 AAATTCCCAC AGGTTGCGGC AACTGGCGAT GGCCCTGACA TTATCTTCTG GGCACACGAC 1801 CGCTTTGGTG GCTACGCTCA ATCTGGCCTG TTGGCTGAAA TCACCCCGGA CAAAGCGTTC 1861 CAGGACAAGC TGTATCCGTT TACCTGGGAT GCCGTACGTT ACAACGGCAA GCTGATTGCT 1921 TACCCGATCG CTGTTGAAGC GTTATCGCTG ATTTATAACA AAGATCTGCT GCCGAACCCG 1981 CCAAAAACCT GGGAAGAGAT CCCGGCGCTG GATAAAGAAC TGAAAGCGAA AGGTAAGAGC 2041 GCGCTGATGT TCAACCTGCA AGAACCGTAC TTCACCTGGC CGCTGATTGC TGCTGACGGG 2101 GGTTATGCGT TCAAGTATGA AAACGGCAAG TACGACATTA AAGACGTGGG CGTGGATAAC 2161 GCTGGCGCGA AAGCGGGTCT GACCTTCCTG GTTGACCTGA TTAAAAACAA ACACATGAAT