pMal-c4X

pMal-c4X

pMalc4X载体基本信息

pMalc4X载体质粒图谱和多克隆位点信息

pMalc4X载体简介

The pMAL™-4 vectors (Figure 1) provide a method for expressing and purifying a protein produced from a cloned gene or open reading frame. The cloned gene is inserted downstream from the malE gene of E. coli, which encodes maltose-binding protein (MBP), resulting in the expression of an MBP fusion protein (1,2). The MBP in these vectors has been engineered for tighter binding to amylose. The method uses the strong “tac” promoter and the malE translation initiation signals to give high-level expression of the cloned sequences (3,4), and a one-step purification of the fusion protein using MBP’s affinity for maltose (5). The vectors express the malE gene (with or without its signal sequence) fused to the lacZα gene. Restriction sites between malE and lacZα are available for inserting the coding sequence of interest. Insertion inactivates the β-galactosidase α-fragment activity of the malE-lacZα fusion, which results in a blue to white color change on Xgal plates when the construction is transformed into an α-complementing host such as TB1, JM107 or NEB 5-alpha Competent E. coli (6,7). When present, the signal peptide on pre-MBP directs fusion proteins to the periplasm. For fusion proteins that can be successfully exported, this allows folding and disulfide bond formation to take place in the periplasm of E. coli, as well as allowing purification of the protein from the periplasm (8). The vectors carry the lacIq gene, which codes for the Lac repressor. This keeps expression from Ptac low in the absence of IPTG induction. The pMAL-4 vectors also contain the sequence coding for the recognition site of a specific protease, located just 5´ to the polylinker insertion sites. This allows MBP to be cleaved from the protein of interest after purification. The pMAL-c4X and pMAL-p4X vectors that are included in the system encode the site for Factor Xa (9, 10). Factor Xa cleaves after its four amino acid recognition sequence, so that few or no vector-derived residues are attached to the protein of interest, depending on the site used for cloning. pMAL vectors containing sites for alternative proteases are also available (Figure 1). The vectors pMAL-c4G (NEB #N8106) and pMAL-p4G (NEB #N8103) encode the site for Genenase™I (NEB #P8075), which cleaves following the sequence His-Tyr. The vectors pMAL-c4E (NEB #N8105) and pMAL-p4E (NEB #N8102) encode the site for Enterokinase (NEB #P8070), which cleaves following the sequence Asp-Asp-Asp-Asp-Lys.

pMalc4X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 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 TTGGTGAAAG ATCCGCGGAT 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 CTCTTTTGTT TATTTTTCTA AATACATTCA AATATGTATC

     3361 CGCTCATGAG ACAATAACCC TGATAAATGC TTCAATAATA TTGAAAAAGG AAGAGTATGA

     3421 GTATTCAACA TTTCCGTGTC GCCCTTATTC CCTTTTTTGC GGCATTTTGC CTTCCTGTTT

     3481 TTGCTCACCC AGAAACGCTG GTGAAAGTAA AAGATGCTGA AGATCAGTTG GGTGCACGAG

     3541 TGGGTTACAT CGAACTGGAT CTCAACAGCG GTAAGATCCT TGAGAGTTTT CGCCCCGAAG

     3601 AACGTTTCCC AATGATGAGC ACTTTTAAAG TTCTGCTATG TGGCGCGGTA TTATCCCGTG

     3661 TTGACGCCGG GCAAGAGCAA CTCGGTCGCC GCATACACTA TTCTCAGAAT GACTTGGTTG

     3721 AGTACTCACC AGTCACAGAA AAGCATCTTA CGGATGGCAT GACAGTAAGA GAATTATGCA

     3781 GTGCTGCCAT AACCATGAGT GATAACACTG CGGCCAACTT ACTTCTGACA ACGATCGGAG

     3841 GACCGAAGGA GCTAACCGCT TTTTTGCACA ACATGGGGGA TCATGTAACT CGCCTTGATC

     3901 GTTGGGAACC GGAGCTGAAT GAAGCCATAC CAAACGACGA GCGTGACACC ACGATGCCTG

     3961 TAGCAATGGC AACAACGTTG CGCAAACTAT TAACTGGCGA ACTACTTACT CTAGCTTCCC

     4021 GGCAACAATT AATAGACTGG ATGGAGGCGG ATAAAGTTGC AGGACCACTT CTGCGCTCGG

     4081 CCCTTCCGGC TGGCTGGTTT ATTGCTGATA AATCTGGAGC CGGTGAGCGT GGGTCTCGCG

     4141 GTATCATTGC AGCACTGGGG CCAGATGGTA AGCCCTCCCG TATCGTAGTT ATCTACACGA

     4201 CGGGGAGTCA GGCAACTATG GATGAACGAA ATAGACAGAT CGCTGAGATA GGTGCCTCAC

     4261 TGATTAAGCA TTGGTAACTG TCAGACCAAG TTTACTCATA TATACTTTAG ATTGATTTAC

     4321 CCCGGTTGAT AATCAGAAAA GCCCCAAAAA CAGGAAGATT GTATAAGCAA ATATTTAAAT

     4381 TGTAAACGTT AATATTTTGT TAAAATTCGC GTTAAATTTT TGTTAAATCA GCTCATTTTT

     4441 TAACCAATAG GCCGAAATCG GCAAAATCCC TTATAAATCA AAAGAATAGA CCGAGATAGG

     4501 GTTGAGTGTT GTTCCAGTTT GGAACAAGAG TCCACTATTA AAGAACGTGG ACTCCAACGT

     4561 CAAAGGGCGA AAAACCGTCT ATCAGGGCGA TGGCCCACTA CGTGAACCAT CACCCAAATC

     4621 AAGTTTTTTG GGGTCGAGGT GCCGTAAAGC ACTAAATCGG AACCCTAAAG GGAGCCCCCG

     4681 ATTTAGAGCT TGACGGGGAA AGCCGGCGAA CGTGGCGAGA AAGGAAGGGA AGAAAGCGAA

     4741 AGGAGCGGGC GCTAGGGCGC TGGCAAGTGT AGCGGTCACG CTGCGCGTAA CCACCACACC

     4801 CGCCGCGCTT AATGCGCCGC TACAGGGCGC GTAAAAGGAT CTAGGTGAAG ATCCTTTTTG

     4861 ATAATCTCAT GACCAAAATC CCTTAACGTG AGTTTTCGTT CCACTGAGCG TCAGACCCCG

     4921 TAGAAAAGAT CAAAGGATCT TCTTGAGATC CTTTTTTTCT GCGCGTAATC TGCTGCTTGC

     4981 AAACAAAAAA ACCACCGCTA CCAGCGGTGG TTTGTTTGCC GGATCAAGAG CTACCAACTC

     5041 TTTTTCCGAA GGTAACTGGC TTCAGCAGAG CGCAGATACC AAATACTGTC CTTCTAGTGT

     5101 AGCCGTAGTT AGGCCACCAC TTCAAGAACT CTGTAGCACC GCCTACATAC CTCGCTCTGC

     5161 TAATCCTGTT ACCAGTGGCT GCTGCCAGTG GCGATAAGTC GTGTCTTACC GGGTTGGACT

     5221 CAAGACGATA GTTACCGGAT AAGGCGCAGC GGTCGGGCTG AACGGGGGGT TCGTGCACAC

     5281 AGCCCAGCTT GGAGCGAACG ACCTACACCG AACTGAGATA CCTACAGCGT GAGCTATGAG

     5341 AAAGCGCCAC GCTTCCCGAA GGGAGAAAGG CGGACAGGTA TCCGGTAAGC GGCAGGGTCG

     5401 GAACAGGAGA GCGCACGAGG GAGCTTCCAG GGGGAAACGC CTGGTATCTT TATAGTCCTG

     5461 TCGGGTTTCG CCACCTCTGA CTTGAGCGTC GATTTTTGTG ATGCTCGTCA GGGGGGCGGA

     5521 GCCTATGGAA AAACGCCAGC AACGCGGCCT TTTTACGGTT CCTGGCCTTT TGCTGGCCTT

     5581 TTGCTCACAT GTTCTTTCCT GCGTTATCCC CTGATTCTGT GGATAACCGT ATTACCGCCT

     5641 TTGAGTGAGC TGATACCGCT CGCCGCAGCC GAACGACCGA GCGCAGCGAG TCAGTGAGCG

     5701 AGGAAGCGGA AGAGCGCCTG ATGCGGTATT TTCTCCTTAC GCATCTGTGC GGTATTTCAC

     5761 ACCGCATATA TGGTGCACTC TCAGTACAAT CTGCTCTGAT GCCGCATAGT TAAGCCAGTA

     5821 TACACTCCGC TATCGCTACG TGACTGGGTC ATGGCTGCGC CCCGACACCC GCCAACACCC

     5881 GCTGACGCGC CCTGACGGGC TTGTCTGCTC CCGGCATCCG CTTACAGACA AGCTGTGACC

     5941 GTCTCCGGGA GCTGCATGTG TCAGAGGTTT TCACCGTCAT CACCGAAACG CGCGAGGCAG

     6001 CTGCGGTAAA GCTCATCAGC GTGGTCGTGC AGCGATTCAC AGATGTCTGC CTGTTCATCC

     6061 GCGTCCAGCT CGTTGAGTTT CTCCAGAAGC GTTAATGTCT GGCTTCTGAT AAAGCGGGCC

     6121 ATGTTAAGGG CGGTTTTTTC CTGTTTGGTC ACTGATGCCT CCGTGTAAGG GGGATTTCTG

     6181 TTCATGGGGG TAATGATACC GATGAAACGA GAGAGGATGC TCACGATACG GGTTACTGAT

     6241 GATGAACATG CCCGGTTACT GGAACGTTGT GAGGGTAAAC AACTGGCGGT ATGGATGCGG

     6301 CGGGACCAGA GAAAAATCAC TCAGGGTCAA TGCCAGCGCT TCGTTAATAC AGATGTAGGT

     6361 GTTCCACAGG GTAGCCAGCA GCATCCTGCG ATGCAGATCC GGAACATAAT GGTGCAGGGC

     6421 GCTGACTTCC GCGTTTCCAG ACTTTACGAA ACACGGAAAC CGAAGACCAT TCATGTTGTT

     6481 GCTCAGGTCG CAGACGTTTT GCAGCAGCAG TCGCTTCACG TTCGCTCGCG TATCGGTGAT

     6541 TCATTCTGCT AACCAGTAAG GCAACCCCGC CAGCCTAGCC GGGTCCTCAA CGACAGGAGC

     6601 ACGATCATGC GCACCCGTGG CCAGGACCCA ACGCTGCCCG AAATT

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其他大肠杆菌表达载体：