pMAL-c2x

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 Binding Protein, MBP）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 system uses the pMAL vectors which are designed so that insertion interrupts a lacZα gene allowing a blue-to-white screen for inserts on X-gal (5). pMAL-c2 series has an exact 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 MBP from the protein of interest, increasing the chances that a particular fusion 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. An EcoR I site in the same reading frame as that of λgt11 and a number of other 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 by infecting with M13KO7 helper phage (NEB #N0315S).

0001 CCGACACCAT CGAATGGTGC AAAACCTTTC GCGGTATGGC ATGATAGCGC CCGGAAGAGA 
0061 GTCAATTCAG GGTGGTGAAT GTGAAACCAG TAACGTTATA CGATGTCGCA GAGTATGCCG 
0121 GTGTCTCTTA TCAGACCGTT TCCCGCGTGG TGAACCAGGC CAGCCACGTT TCTGCGAAAA 
0181 CGCGGGAAAA AGTGGAAGCG GCGATGGCGG AGCTGAATTA CATTCCCAAC CGCGTGGCAC 
0241 AACAACTGGC GGGCAAACAG TCGTTGCTGA TTGGCGTTGC CACCTCCAGT CTGGCCCTGC 
0301 ACGCGCCGTC GCAAATTGTC GCGGCGATTA AATCTCGCGC CGATCAACTG GGTGCCAGCG 
0361 TGGTGGTGTC GATGGTAGAA CGAAGCGGCG TCGAAGCCTG TAAAGCGGCG GTGCACAATC 
0421 TTCTCGCGCA ACGCGTCAGT GGGCTGATCA TTAACTATCC GCTGGATGAC CAGGATGCCA 
0481 TTGCTGTGGA AGCTGCCTGC ACTAATGTTC CGGCGTTATT TCTTGATGTC TCTGACCAGA 
0541 CACCCATCAA CAGTATTATT TTCTCCCATG AAGACGGTAC GCGACTGGGC GTGGAGCATC 
0601 TGGTCGCATT GGGTCACCAG CAAATCGCGC TGTTAGCGGG CCCATTAAGT TCTGTCTCGG 
0661 CGCGTCTGCG TCTGGCTGGC TGGCATAAAT ATCTCACTCG CAATCAAATT CAGCCGATAG 
0721 CGGAACGGGA AGGCGACTGG AGTGCCATGT CCGGTTTTCA ACAAACCATG CAAATGCTGA 
0781 ATGAGGGCAT CGTTCCCACT GCGATGCTGG TTGCCAACGA TCAGATGGCG CTGGGCGCAA 
0841 TGCGCGCCAT TACCGAGTCC GGGCTGCGCG TTGGTGCGGA TATCTCGGTA GTGGGATACG 
0901 ACGATACCGA AGACAGCTCA TGTTATATCC CGCCGTTAAC CACCATCAAA CAGGATTTTC 
0961 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 AAAATCGAAG AAGGTAAACT GGTAATCTGG 
1561 ATTAACGGCG ATAAAGGCTA TAACGGTCTC GCTGAAGTCG GTAAGAAATT CGAGAAAGAT 
1621 ACCGGAATTA AAGTCACCGT TGAGCATCCG GATAAACTGG AAGAGAAATT CCCACAGGTT 
1681 GCGGCAACTG GCGATGGCCC TGACATTATC TTCTGGGCAC ACGACCGCTT TGGTGGCTAC 
1741 GCTCAATCTG GCCTGTTGGC TGAAATCACC CCGGACAAAG CGTTCCAGGA CAAGCTGTAT 
1801 CCGTTTACCT GGGATGCCGT ACGTTACAAC GGCAAGCTGA TTGCTTACCC GATCGCTGTT 
1861 GAAGCGTTAT CGCTGATTTA TAACAAAGAT CTGCTGCCGA ACCCGCCAAA AACCTGGGAA 
1921 GAGATCCCGG CGCTGGATAA AGAACTGAAA GCGAAAGGTA AGAGCGCGCT GATGTTCAAC 
1981 CTGCAAGAAC CGTACTTCAC CTGGCCGCTG ATTGCTGCTG ACGGGGGTTA TGCGTTCAAG 
2041 TATGAAAACG GCAAGTACGA CATTAAAGAC GTGGGCGTGG ATAACGCTGG CGCGAAAGCG 
2101 GGTCTGACCT TCCTGGTTGA CCTGATTAAA AACAAACACA TGAATGCAGA CACCGATTAC 
2161 TCCATCGCAG AAGCTGCCTT TAATAAAGGC GAAACAGCGA TGACCATCAA CGGCCCGTGG 
2221 GCATGGTCCA ACATCGACAC CAGCAAAGTG AATTATGGTG TAACGGTACT GCCGACCTTC 
2281 AAGGGTCAAC CATCCAAACC GTTCGTTGGC GTGCTGAGCG CAGGTATTAA CGCCGCCAGT 
2341 CCGAACAAAG AGCTGGCAAA AGAGTTCCTC GAAAACTATC TGCTGACTGA TGAAGGTCTG 
2401 GAAGCGGTTA ATAAAGACAA ACCGCTGGGT GCCGTAGCGC TGAAGTCTTA CGAGGAAGAG 
2461 TTGGCGAAAG ATCCACGTAT TGCCGCCACT ATGGAAAACG CCCAGAAAGG TGAAATCATG 
2521 CCGAACATCC CGCAGATGTC CGCTTTCTGG TATGCCGTGC GTACTGCGGT GATCAACGCC 
2581 GCCAGCGGTC GTCAGACTGT CGATGAAGCC CTGAAAGACG CGCAGACTAA TTCGAGCTCG 
2641 AACAACAACA ACAATAACAA TAACAACAAC CTCGGGATCG AGGGAAGGAT TTCAGAATTC 
2701 GGATCCTCTA GAGTCGACCT GCAGGCAAGC TTGGCACTGG CCGTCGTTTT ACAACGTCGT 
2761 GACTGGGAAA ACCCTGGCGT TACCCAACTT AATCGCCTTG CAGCACATCC CCCTTTCGCC 
2821 AGCTGGCGTA ATAGCGAAGA GGCCCGCACC GATCGCCCTT CCCAACAGTT GCGCAGCCTG 
2881 AATGGCGAAT GGCAGCTTGG CTGTTTTGGC GGATGAGATA AGATTTTCAG CCTGATACAG 
2941 ATTAAATCAG AACGCAGAAG CGGTCTGATA AAACAGAATT TGCCTGGCGG CAGTAGCGCG 
3001 GTGGTCCCAC CTGACCCCAT GCCGAACTCA GAAGTGAAAC GCCGTAGCGC CGATGGTAGT 
3061 GTGGGGTCTC CCCATGCGAG AGTAGGGAAC TGCCAGGCAT CAAATAAAAC GAAAGGCTCA 
3121 GTCGAAAGAC TGGGCCTTTC GTTTTATCTG TTGTTTGTCG GTGAACGCTC TCCTGAGTAG 
3181 GACAAATCCG CCGGGAGCGG ATTTGAACGT TGCGAAGCAA CGGCCCGGAG GGTGGCGGGC 
3241 AGGACGCCCG CCATAAACTG CCAGGCATCA AATTAAGCAG AAGGCCATCC TGACGGATGG 
3301 CCTTTTTGCG TTTCTACAAA CTCTTTTTGT TTATTTTTCT AAATACATTC AAATATGTAT 
3361 CCGCTCATGA GACAATAACC CTGATAAATG CTTCAATAAT ATTGAAAAAG GAAGAGTATG 
3421 AGTATTCAAC ATTTCCGTGT CGCCCTTATT CCCTTTTTTG CGGCATTTTG CCTTCCTGTT 
3481 TTTGCTCACC CAGAAACGCT GGTGAAAGTA AAAGATGCTG AAGATCAGTT GGGTGCACGA 
3541 GTGGGTTACA TCGAACTGGA TCTCAACAGC GGTAAGATCC TTGAGAGTTT TCGCCCCGAA 
3601 GAACGTTCTC CAATGATGAG CACTTTTAAA GTTCTGCTAT GTGGCGCGGT ATTATCCCGT 
3661 GTTGACGCCG GGCAAGAGCA ACTCGGTCGC CGCATACACT ATTCTCAGAA TGACTTGGTT 
3721 GAGTACTCAC CAGTCACAGA AAAGCATCTT ACGGATGGCA TGACAGTAAG AGAATTATGC 
3781 AGTGCTGCCA TAACCATGAG TGATAACACT GCGGCCAACT TACTTCTGAC AACGATCGGA 
3841 GGACCGAAGG AGCTAACCGC TTTTTTGCAC AACATGGGGG ATCATGTAAC TCGCCTTGAT 
3901 CGTTGGGAAC CGGAGCTGAA TGAAGCCATA CCAAACGACG AGCGTGACAC CACGATGCCT 
3961 GTAGCAATGG CAACAACGTT GCGCAAACTA TTAACTGGCG AACTACTTAC TCTAGCTTCC 
4021 CGGCAACAAT TAATAGACTG GATGGAGGCG GATAAAGTTG CAGGACCACT TCTGCGCTCG 
4081 GCCCTTCCGG CTGGCTGGTT TATTGCTGAT AAATCTGGAG CCGGTGAGCG TGGGTCTCGC 
4141 GGTATCATTG CAGCACTGGG GCCAGATGGT AAGCCCTCCC GTATCGTAGT TATCTACACG 
4201 ACGGGGAGTC AGGCAACTAT GGATGAACGA AATAGACAGA TCGCTGAGAT AGGTGCCTCA 
4261 CTGATTAAGC ATTGGTAACT GTCAGACCAA GTTTACTCAT ATATACTTTA GATTGATTTA 
4321 CCCCGGTTGA TAATCAGAAA AGCCCCAAAA ACAGGAAGAT TGTATAAGCA AATATTTAAA 
4381 TTGTAAACGT TAATATTTTG TTAAAATTCG CGTTAAATTT TTGTTAAATC AGCTCATTTT 
4441 TTAACCAATA GGCCGAAATC GGCAAAATCC CTTATAAATC AAAAGAATAG ACCGAGATAG 
4501 GGTTGAGTGT TGTTCCAGTT TGGAACAAGA GTCCACTATT AAAGAACGTG GACTCCAACG 
4561 TCAAAGGGCG AAAAACCGTC TATCAGGGCG ATGGCCCACT ACGTGAACCA TCACCCAAAT 
4621 CAAGTTTTTT GGGGTCGAGG TGCCGTAAAG CACTAAATCG GAACCCTAAA GGGAGCCCCC 
4681 GATTTAGAGC TTGACGGGGA AAGCCGGCGA ACGTGGCGAG AAAGGAAGGG AAGAAAGCGA 
4741 AAGGAGCGGG CGCTAGGGCG CTGGCAAGTG TAGCGGTCAC GCTGCGCGTA ACCACCACAC 
4801 CCGCCGCGCT TAATGCGCCG CTACAGGGCG CGTAAAAGGA TCTAGGTGAA GATCCTTTTT 
4861 GATAATCTCA TGACCAAAAT CCCTTAACGT GAGTTTTCGT TCCACTGAGC GTCAGACCCC 
4921 GTAGAAAAGA TCAAAGGATC TTCTTGAGAT CCTTTTTTTC TGCGCGTAAT CTGCTGCTTG 
4981 CAAACAAAAA AACCACCGCT ACCAGCGGTG GTTTGTTTGC CGGATCAAGA GCTACCAACT 
5041 CTTTTTCCGA AGGTAACTGG CTTCAGCAGA GCGCAGATAC CAAATACTGT CCTTCTAGTG 
5101 TAGCCGTAGT TAGGCCACCA CTTCAAGAAC TCTGTAGCAC CGCCTACATA CCTCGCTCTG 
5161 CTAATCCTGT TACCAGTGGC TGCTGCCAGT GGCGATAAGT CGTGTCTTAC CGGGTTGGAC 
5221 TCAAGACGAT AGTTACCGGA TAAGGCGCAG CGGTCGGGCT GAACGGGGGG TTCGTGCACA 
5281 CAGCCCAGCT TGGAGCGAAC GACCTACACC GAACTGAGAT ACCTACAGCG TGAGCTATGA 
5341 GAAAGCGCCA CGCTTCCCGA AGGGAGAAAG GCGGACAGGT ATCCGGTAAG CGGCAGGGTC 
5401 GGAACAGGAG AGCGCACGAG GGAGCTTCCA GGGGGAAACG CCTGGTATCT TTATAGTCCT 
5461 GTCGGGTTTC GCCACCTCTG ACTTGAGCGT CGATTTTTGT GATGCTCGTC AGGGGGGCGG 
5521 AGCCTATGGA AAAACGCCAG CAACGCGGCC TTTTTACGGT TCCTGGCCTT TTGCTGGCCT 
5581 TTTGCTCACA TGTTCTTTCC TGCGTTATCC CCTGATTCTG TGGATAACCG TATTACCGCC 
5641 TTTGAGTGAG CTGATACCGC TCGCCGCAGC CGAACGACCG AGCGCAGCGA GTCAGTGAGC 
5701 GAGGAAGCGG AAGAGCGCCT GATGCGGTAT TTTCTCCTTA CGCATCTGTG CGGTATTTCA 
5761 CACCGCATAT ATGGTGCACT CTCAGTACAA TCTGCTCTGA TGCCGCATAG TTAAGCCAGT 
5821 ATACACTCCG CTATCGCTAC GTGACTGGGT CATGGCTGCG CCCCGACACC CGCCAACACC 
5881 CGCTGACGCG CCCTGACGGG CTTGTCTGCT CCCGGCATCC GCTTACAGAC AAGCTGTGAC 
5941 CGTCTCCGGG AGCTGCATGT GTCAGAGGTT TTCACCGTCA TCACCGAAAC GCGCGAGGCA 
6001 GCTGCGGTAA AGCTCATCAG CGTGGTCGTG CAGCGATTCA CAGATGTCTG CCTGTTCATC 
6061 CGCGTCCAGC TCGTTGAGTT TCTCCAGAAG CGTTAATGTC TGGCTTCTGA TAAAGCGGGC 
6121 CATGTTAAGG GCGGTTTTTT CCTGTTTGGT CACTGATGCC TCCGTGTAAG GGGGATTTCT 
6181 GTTCATGGGG GTAATGATAC CGATGAAACG AGAGAGGATG CTCACGATAC GGGTTACTGA 
6241 TGATGAACAT GCCCGGTTAC TGGAACGTTG TGAGGGTAAA CAACTGGCGG TATGGATGCG 
6301 GCGGGACCAG AGAAAAATCA CTCAGGGTCA ATGCCAGCGC TTCGTTAATA CAGATGTAGG 
6361 TGTTCCACAG GGTAGCCAGC AGCATCCTGC GATGCAGATC CGGAACATAA TGGTGCAGGG 
6421 CGCTGACTTC CGCGTTTCCA GACTTTACGA AACACGGAAA CCGAAGACCA TTCATGTTGT 
6481 TGCTCAGGTC GCAGACGTTT TGCAGCAGCA GTCGCTTCAC GTTCGCTCGC GTATCGGTGA 
6541 TTCATTCTGC TAACCAGTAA GGCAACCCCG CCAGCCTAGC CGGGTCCTCA ACGACAGGAG 
6601 CACGATCATG CGCACCCGTG GCCAGGACCC AACGCTGCCC GAAATT