pMAL-p4x

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.

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 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 
2221 GCAGACACCG ATTACTCCAT CGCAGAAGCT GCCTTTAATA AAGGCGAAAC AGCGATGACC 
2281 ATCAACGGCC CGTGGGCATG GTCCAACATC GACACCAGCA AAGTGAATTA TGGTGTAACG 
2341 GTACTGCCGA CCTTCAAGGG TCAACCATCC AAACCGTTCG TTGGCGTGCT GAGCGCAGGT 
2401 ATTAACGCCG CCAGTCCGAA CAAAGAGCTG GCAAAAGAGT TCCTCGAAAA CTATCTGCTG 
2461 ACTGATGAAG GTCTGGAAGC GGTTAATAAA GACAAACCGC TGGGTGCCGT AGCGCTGAAG 
2521 TCTTACGAGG AAGAGTTGGT GAAAGATCCG CGGATTGCCG CCACTATGGA AAACGCCCAG 
2581 AAAGGTGAAA TCATGCCGAA CATCCCGCAG ATGTCCGCTT TCTGGTATGC CGTGCGTACT 
2641 GCGGTGATCA ACGCCGCCAG CGGTCGTCAG ACTGTCGATG AAGCCCTGAA AGACGCGCAG 
2701 ACTAATTCGA GCTCGAACAA CAACAACAAT AACAATAACA ACAACCTCGG GATCGAGGGA 
2761 AGGATTTCAG AATTCGGATC CTCTAGAGTC GACCTGCAGG CAAGCTTGGC ACTGGCCGTC 
2821 GTTTTACAAC GTCGTGACTG GGAAAACCCT GGCGTTACCC AACTTAATCG CCTTGCAGCA 
2881 CATCCCCCTT TCGCCAGCTG GCGTAATAGC GAAGAGGCCC GCACCGATCG CCCTTCCCAA 
2941 CAGTTGCGCA GCCTGAATGG CGAATGGCAG CTTGGCTGTT TTGGCGGATG AGATAAGATT 
3001 TTCAGCCTGA TACAGATTAA ATCAGAACGC AGAAGCGGTC TGATAAAACA GAATTTGCCT 
3061 GGCGGCAGTA GCGCGGTGGT CCCACCTGAC CCCATGCCGA ACTCAGAAGT GAAACGCCGT 
3121 AGCGCCGATG GTAGTGTGGG GTCTCCCCAT GCGAGAGTAG GGAACTGCCA GGCATCAAAT 
3181 AAAACGAAAG GCTCAGTCGA AAGACTGGGC CTTTCGTTTT ATCTGTTGTT TGTCGGTGAA 
3241 CGCTCTCCTG AGTAGGACAA ATCCGCCGGG AGCGGATTTG AACGTTGCGA AGCAACGGCC 
3301 CGGAGGGTGG CGGGCAGGAC GCCCGCCATA AACTGCCAGG CATCAAATTA AGCAGAAGGC 
3361 CATCCTGACG GATGGCCTTT TTGCGTTTCT ACAAACTCTT TTGTTTATTT TTCTAAATAC 
3421 ATTCAAATAT GTATCCGCTC ATGAGACAAT AACCCTGATA AATGCTTCAA TAATATTGAA 
3481 AAAGGAAGAG TATGAGTATT CAACATTTCC GTGTCGCCCT TATTCCCTTT TTTGCGGCAT 
3541 TTTGCCTTCC TGTTTTTGCT CACCCAGAAA CGCTGGTGAA AGTAAAAGAT GCTGAAGATC 
3601 AGTTGGGTGC ACGAGTGGGT TACATCGAAC TGGATCTCAA CAGCGGTAAG ATCCTTGAGA 
3661 GTTTTCGCCC CGAAGAACGT TTCCCAATGA TGAGCACTTT TAAAGTTCTG CTATGTGGCG 
3721 CGGTATTATC CCGTGTTGAC GCCGGGCAAG AGCAACTCGG TCGCCGCATA CACTATTCTC 
3781 AGAATGACTT GGTTGAGTAC TCACCAGTCA CAGAAAAGCA TCTTACGGAT GGCATGACAG 
3841 TAAGAGAATT ATGCAGTGCT GCCATAACCA TGAGTGATAA CACTGCGGCC AACTTACTTC 
3901 TGACAACGAT CGGAGGACCG AAGGAGCTAA CCGCTTTTTT GCACAACATG GGGGATCATG 
3961 TAACTCGCCT TGATCGTTGG GAACCGGAGC TGAATGAAGC CATACCAAAC GACGAGCGTG 
4021 ACACCACGAT GCCTGTAGCA ATGGCAACAA CGTTGCGCAA ACTATTAACT GGCGAACTAC 
4081 TTACTCTAGC TTCCCGGCAA CAATTAATAG ACTGGATGGA GGCGGATAAA GTTGCAGGAC 
4141 CACTTCTGCG CTCGGCCCTT CCGGCTGGCT GGTTTATTGC TGATAAATCT GGAGCCGGTG 
4201 AGCGTGGGTC TCGCGGTATC ATTGCAGCAC TGGGGCCAGA TGGTAAGCCC TCCCGTATCG 
4261 TAGTTATCTA CACGACGGGG AGTCAGGCAA CTATGGATGA ACGAAATAGA CAGATCGCTG 
4321 AGATAGGTGC CTCACTGATT AAGCATTGGT AACTGTCAGA CCAAGTTTAC TCATATATAC 
4381 TTTAGATTGA TTTACCCCGG TTGATAATCA GAAAAGCCCC AAAAACAGGA AGATTGTATA 
4441 AGCAAATATT TAAATTGTAA ACGTTAATAT TTTGTTAAAA TTCGCGTTAA ATTTTTGTTA 
4501 AATCAGCTCA TTTTTTAACC AATAGGCCGA AATCGGCAAA ATCCCTTATA AATCAAAAGA 
4561 ATAGACCGAG ATAGGGTTGA GTGTTGTTCC AGTTTGGAAC AAGAGTCCAC TATTAAAGAA 
4621 CGTGGACTCC AACGTCAAAG GGCGAAAAAC CGTCTATCAG GGCGATGGCC CACTACGTGA 
4681 ACCATCACCC AAATCAAGTT TTTTGGGGTC GAGGTGCCGT AAAGCACTAA ATCGGAACCC 
4741 TAAAGGGAGC CCCCGATTTA GAGCTTGACG GGGAAAGCCG GCGAACGTGG CGAGAAAGGA 
4801 AGGGAAGAAA GCGAAAGGAG CGGGCGCTAG GGCGCTGGCA AGTGTAGCGG TCACGCTGCG 
4861 CGTAACCACC ACACCCGCCG CGCTTAATGC GCCGCTACAG GGCGCGTAAA AGGATCTAGG 
4921 TGAAGATCCT TTTTGATAAT CTCATGACCA AAATCCCTTA ACGTGAGTTT TCGTTCCACT 
4981 GAGCGTCAGA CCCCGTAGAA AAGATCAAAG GATCTTCTTG AGATCCTTTT TTTCTGCGCG 
5041 TAATCTGCTG CTTGCAAACA AAAAAACCAC CGCTACCAGC GGTGGTTTGT TTGCCGGATC 
5101 AAGAGCTACC AACTCTTTTT CCGAAGGTAA CTGGCTTCAG CAGAGCGCAG ATACCAAATA 
5161 CTGTCCTTCT AGTGTAGCCG TAGTTAGGCC ACCACTTCAA GAACTCTGTA GCACCGCCTA 
5221 CATACCTCGC TCTGCTAATC CTGTTACCAG TGGCTGCTGC CAGTGGCGAT AAGTCGTGTC 
5281 TTACCGGGTT GGACTCAAGA CGATAGTTAC CGGATAAGGC GCAGCGGTCG GGCTGAACGG 
5341 GGGGTTCGTG CACACAGCCC AGCTTGGAGC GAACGACCTA CACCGAACTG AGATACCTAC 
5401 AGCGTGAGCT ATGAGAAAGC GCCACGCTTC CCGAAGGGAG AAAGGCGGAC AGGTATCCGG 
5461 TAAGCGGCAG GGTCGGAACA GGAGAGCGCA CGAGGGAGCT TCCAGGGGGA AACGCCTGGT 
5521 ATCTTTATAG TCCTGTCGGG TTTCGCCACC TCTGACTTGA GCGTCGATTT TTGTGATGCT 
5581 CGTCAGGGGG GCGGAGCCTA TGGAAAAACG CCAGCAACGC GGCCTTTTTA CGGTTCCTGG 
5641 CCTTTTGCTG GCCTTTTGCT CACATGTTCT TTCCTGCGTT ATCCCCTGAT TCTGTGGATA 
5701 ACCGTATTAC CGCCTTTGAG TGAGCTGATA CCGCTCGCCG CAGCCGAACG ACCGAGCGCA 
5761 GCGAGTCAGT GAGCGAGGAA GCGGAAGAGC GCCTGATGCG GTATTTTCTC CTTACGCATC 
5821 TGTGCGGTAT TTCACACCGC ATATATGGTG CACTCTCAGT ACAATCTGCT CTGATGCCGC 
5881 ATAGTTAAGC CAGTATACAC TCCGCTATCG CTACGTGACT GGGTCATGGC TGCGCCCCGA 
5941 CACCCGCCAA CACCCGCTGA CGCGCCCTGA CGGGCTTGTC TGCTCCCGGC ATCCGCTTAC 
6001 AGACAAGCTG TGACCGTCTC CGGGAGCTGC ATGTGTCAGA GGTTTTCACC GTCATCACCG 
6061 AAACGCGCGA GGCAGCTGCG GTAAAGCTCA TCAGCGTGGT CGTGCAGCGA TTCACAGATG 
6121 TCTGCCTGTT CATCCGCGTC CAGCTCGTTG AGTTTCTCCA GAAGCGTTAA TGTCTGGCTT 
6181 CTGATAAAGC GGGCCATGTT AAGGGCGGTT TTTTCCTGTT TGGTCACTGA TGCCTCCGTG 
6241 TAAGGGGGAT TTCTGTTCAT GGGGGTAATG ATACCGATGA AACGAGAGAG GATGCTCACG 
6301 ATACGGGTTA CTGATGATGA ACATGCCCGG TTACTGGAAC GTTGTGAGGG TAAACAACTG 
6361 GCGGTATGGA TGCGGCGGGA CCAGAGAAAA ATCACTCAGG GTCAATGCCA GCGCTTCGTT 
6421 AATACAGATG TAGGTGTTCC ACAGGGTAGC CAGCAGCATC CTGCGATGCA GATCCGGAAC 
6481 ATAATGGTGC AGGGCGCTGA CTTCCGCGTT TCCAGACTTT ACGAAACACG GAAACCGAAG 
6541 ACCATTCATG TTGTTGCTCA GGTCGCAGAC GTTTTGCAGC AGCAGTCGCT TCACGTTCGC 
6601 TCGCGTATCG GTGATTCATT CTGCTAACCA GTAAGGCAAC CCCGCCAGCC TAGCCGGGTC 
6661 CTCAACGACA GGAGCACGAT CATGCGCACC CGTGGCCAGG ACCCAACGCT GCCCGAAATT