pMAL-p5E

The vector pMAL-p5E is designed to produce maltose-binding protein (MBP) fusions, where the protein of interest can be cleaved from MBP with the specific protease Enterokinase (NEB #P8070).

MBP fusions made with this vector include an N-terminal signal sequence, so the fusion protein is directed to the periplasm. The MBP has been engineered for tigher binding to amylose resin.

A gene or open reading frame is inserted into a restriction site of the vector polylinker, in the same translational reading frame as the malE gene (encoding MBP). The fusion protein produced from the vector can be purified by amylose affinity chromatography. The sequence coding for the five amino acids Asp-Asp-Asp-Asp-Lys is present just upstream of the KpnI site. This allows the protein of interest to be cleaved from maltose-binding protein with enterokinase.

pMAL-p5E cut with KpnI followed by treatment with the Quick Blunting Kit (NEB #E1201) produces a blunt end at the lysine codon. This allows blunt-end cloning of an insert where the first three nucleotides code for the first amino acid of the protein of interest, and enterokinase cleavage of the fusion produces a protein with no vector-derived amino acids.

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 TATTTCGGTA 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 GCAATTGACC AACAAGGACC ATAGATTATG 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 CGTATTGCCG CCACTATGGA AAACGCCCAG 
2581 AAAGGTGAAA TCATGCCGAA CATCCCGCAG ATGTCCGCTT TCTGGTATGC CGTGCGTACT 
2641 GCGGTGATCA ACGCCGCCAG CGGTCGTCAG ACTGTCGATG AAGCCCTGAA AGACGCGCAG 
2701 ACTAATTCGA GCTCGAACAA CAACAACAAT AACAATAACA ACAACCTCGG GGATGACGAT 
2761 GACAAGGTAC CGCATATGTC CATGGGCGGC CGCGATATCG TCGACGGATC CGAATTCCCT 
2821 GCAGGTAATT AAATAAGCTT CAAATAAAAC GAAAGGCTCA GTCGAAAGAC TGGGCCTTTC 
2881 GTTTTATCTG TTGTTTGTCG GTGAACGCTC TCCTGAGTAG GACAAATCCG CCGGGAGCGG 
2941 ATTTGAACGT TGCGAAGCAA CGGCCCGGAG GGTGGCGGGC AGGACGCCCG CCATAAACTG 
3001 CCAGGCATCA AATTAAGCAG AAGGCCATCC TGACGGATGG CCTTTTTGCG TTTCTACAAA 
3061 CTCTTTCGGT CCGTTGTTTA TTTTTCTAAA TACATTCAAA TATGTATCCG CTCATGAGAC 
3121 AATAACCCTG ATAAATGCTT CAATAATATT GAAAAAGGAA GAGTATGAGT ATTCAACATT 
3181 TCCGTGTCGC CCTTATTCCC TTTTTTGCGG CATTTTGCCT TCCTGTTTTT GCTCACCCAG 
3241 AAACGCTGGT GAAAGTAAAA GATGCTGAAG ATCAGTTGGG TGCACGAGTG GGTTACATCG 
3301 AACTGGATCT CAACAGCGGT AAGATCCTTG AGAGTTTTCG CCCCGAAGAA CGTTTCCCAA 
3361 TGATGAGCAC TTTTAAAGTT CTGCTATGTG GCGCGGTATT ATCCCGTGTT GACGCCGGGC 
3421 AAGAGCAACT CGGTCGCCGC ATACACTATT CTCAGAATGA CTTGGTTGAG TACTCACCAG 
3481 TCACAGAAAA GCATCTTACG GATGGCATGA CAGTAAGAGA ATTATGCAGT GCTGCCATAA 
3541 CCATGAGTGA TAACACTGCG GCCAACTTAC TTCTGACAAC GATCGGAGGA CCGAAGGAGC 
3601 TAACCGCTTT TTTGCACAAC ATGGGGGATC ATGTAACTCG CCTTGATCGT TGGGAACCGG 
3661 AGCTGAATGA AGCCATACCA AACGACGAGC GTGACACCAC GATGCCTGTA GCAATGGCAA 
3721 CAACGTTGCG CAAACTATTA ACTGGCGAAC TACTTACTCT AGCTTCCCGG CAACAATTAA 
3781 TAGACTGGAT GGAGGCGGAT AAAGTTGCAG GACCACTTCT GCGCTCGGCC CTTCCGGCTG 
3841 GCTGGTTTAT TGCTGATAAA TCTGGAGCCG GTGAGCGTGG GTCTCGCGGT ATCATTGCAG 
3901 CACTGGGGCC AGATGGTAAG CCCTCCCGTA TCGTAGTTAT CTACACGACG GGGAGTCAGG 
3961 CAACTATGGA TGAACGAAAT AGACAGATCG CTGAGATAGG TGCCTCACTG ATTAAGCATT 
4021 GGTAACTGTC AGACCAAGTT TACTCATATA TACTTTAGAT TGATTTCCTT AGGACTGAGC 
4081 GTCAACCCCG TAGAAAAGAT CAAAGGATCT TCTTGAGATC CTTTTTTTCT GCGCGTAATC 
4141 TGCTGCTTGC AAACAAAAAA ACCACCGCTA CCAGCGGTGG TTTGTTTGCC GGATCAAGAG 
4201 CTACCAACTC TTTTTCCGAA GGTAACTGGC TTCAGCAGAG CGCAGATACC AAATACTGTC 
4261 CTTCTAGTGT AGCCGTAGTT AGGCCACCAC TTCAAGAACT CTGTAGCACC GCCTACATAC 
4321 CTCGCTCTGC TAATCCTGTT ACCAGTGGCT GCTGCCAGTG GCGATAAGTC GTGTCTTACC 
4381 GGGTTGGACT CAAGACGATA GTTACCGGAT AAGGCGCAGC GGTCGGGCTG AACGGGGGGT 
4441 TCGTGCACAC AGCCCAGCTT GGAGCGAACG ACCTACACCG AACTGAGATA CCTACAGCGT 
4501 GAGCTATGAG AAAGCGCCAC GCTTCCCGAA GGGAGAAAGG CGGACAGGTA TCCGGTAAGC 
4561 GGCAGGGTCG GAACAGGAGA GCGCACGAGG GAGCTTCCAG GGGGAAACGC CTGGTATCTT 
4621 TATAGTCCTG TCGGGTTTCG CCACCTCTGA CTTGAGCGTC GATTTTTGTG ATGCTCGTCA 
4681 GGGGGGCGGA GCCTATGGAA AAACGCCAGC AACGCGGCCT TTTTACGGTT CCTGGCCTTT 
4741 TGCTGGCCTT TTGCTCACAT GTTCTTTCCT GCGTTATCCC CTGATTCTGT GGATAACCGT 
4801 ATTACCGCCT TTGAGTGAGC TGATACCGCT CGCCGCAGCC GAACGACCGA GCGCAGCGAG 
4861 TCAGTGAGCG AGGAAGCGGA AGAGCGCCTG ATGCGGTATT TTCTCCTTAC GCATCTGTGC 
4921 GGTATTTCAC ACCGCATATA AGGTGCACTG TGACTGGGTC ATGGCTGCGC CCCGACACCC 
4981 GCCAACACCC GCTGACGCGC CCTGACGGGC TTGTCTGCTC CCGGCATCCG CTTACAGACA 
5041 AGCTGTGACC GTCTCCGGGA GCTGCATGTG TCAGAGGTTT TCACCGTCAT CACCGAAACG 
5101 CGCGAGGCAG CTGCGGTAAA GCTCATCAGC GTGGTCGTGC AGCGATTCAC AGATGTCTGC 
5161 CTGTTCATCC GCGTCCAGCT CGTTGAGTTT CTCCAGAAGC GTTAATGTCT GGCTTCTGAT 
5221 AAAGCGGGCC ATGTTAAGGG CGGTTTTTTC CTGTTTGGTC ACTGATGCCT CCGTGTAAGG 
5281 GGGATTTCTG TTCATGGGGG TAATGATACC GATGAAACGA GAGAGGATGC TCACGATACG 
5341 GGTTACTGAT GATGAACATG CCCGGTTACT GGAACGTTGT GAGGGTAAAC AACTGGCGGT 
5401 ATGGATGCGG CGGGACCAGA GAAAAATCAC TCAGGGTCAA TGCCAGCGCT TCGTTAATAC 
5461 AGATGTAGGT GTTCCACAGG GTAGCCAGCA GCATCCTGCG ATGCAGATCC GGAACATAAT 
5521 GGTGCAGGGC GCTGACTTCC GCGTTTCCAG ACTTTACGAA ACACGGAAAC CGAAGACCAT 
5581 TCATGTTGTT GCTCAGGTCG CAGACGTTTT GCAGCAGCAG TCGCTTCACG TTCGCTCGCG 
5641 TATCGGTGAT TCATTCTGCT AACCAGTAAG GCAACCCCGC CAGCCTAGCC GGGTCCTCAA 
5701 CGACAGGAGC ACGATCATGC GCACCCGTGG CCAGGACCCA ACGCTGCCCG AAATT