Gaffney notes, May 26, 2015
Soybean lipoxygenase-1 (similar to gb|AAA33986.1|, 839 aa) was cloned into pET-30 Xa/LIC. The sequence of the expression construct (below) is numbered beginning with the ATG start of the protein-coding region.The mRNA (approx. 1.8 mg from 1 g seedpods)was obtained from green pods of plants grown from soybeans packaged by the former Glacial Ridge Foods, Starbuck, MN.Intermediate steps in amplifying the cDNA are described in a dissertation by Brendan C. Maguire (Johns Hopkins University, 1999, UMI Microform 9927119)1. The coding cDNA sequence differed from published in bases 478-480 (AGT to GAG, Ser160Glu) and at base 1608 (G to T, Lys640, silent) (Coffa et al. 2005, J. Biol. Chem. 280: 38756)2. Ligation independent cloning was used to insert the lipoxygenase gene into pET-30 Xa/LIC, to give a construct of 7978 bp, with 2517 bp of lipoxygenase gene followed by an 11 bp C-terminal extension and then 5444 bp of the vector. The C-terminal extension includes a stop codon and a SpeI restriction site (underlined), (TAAACTAGTCATGGGGC)and it renders the C-terminal His-tag codons in the vector out of frame. Minor variations from the published sequence of the pET-30 Xa vector occur at positions 3224 (T to C), 3234 (C deletion), and 5087-5089 (TAT deletion). The same changes have also been found in a pET 28 plasmid, suggesting these are common variants of pET vectors.3
The deposited plasmids include the wild-type protein sequence as two variants: WT160E and WT160S, both with the pETXa30Xa/LIC N-terminal His-tag appended. Plasmids for His-tagged lipoxygenases in which the four native cysteines codons have been mutated to serine (TGC to TCC) are included for both His-tagged WT sequences (160E and 160S). Wild-type constructs, 160E and 160S, lacking the 141 bp N-terminal extension coding for the pET-30Xa His tag were also prepared using Q5 mutagenesis (NEB)3.
REFERENCES
1.Brendan C. Maguire, Dissertation, Johns Hopkins University, 1999, UMI Microform: 9927119.
2. G. Coffa, A.N. Imber, B.C. Maguire, G. Laxmikanthan, C. Schneider, B.J. Gaffney, and A.R. Brash (2005) "On the Relationships of Substrate Orientation, Hydrogen Abstraction and Product Stereochemistry in Single and Double Dioxygenations by Soybean Lipoxygenase-1 and Its Ala542Gly Mutant" J. Biol. Chem. 280: 38756-38766.
3. B.C. Maguire, F. Wu, B. Washburn and B.J. Gaffney, unpublished.
SEQUENCE
1ATGTTTTCAG CAGGCCATAA GATCAAAGGG ACAGTGGTGT TGATGCCCAA GAATGAGTTG
61 GAAGTTAACC CTGATGGCTC AGCAGTTGAC AACCTTAATG CTTTCTTGGG CCGTAGTGTC
121 TCCCTTCAGC TCATTAGTGC TACCAAGGCT GATGCACATG GAAAAGGAAA AGTTGGAAAG
181 GATACGTTCT TGGAAGGGAT TAATACTTCG TTACCAACTT TGGGAGCAGG AGAGTCTGCA
241 TTCAATATTC ATTTTGAATG GGACGGAAGC ATGGGAATCC CCGGTGCGTT TTACATAAAG
301 AACTACATGC AAGTTGAGTT CTTCCTCAAG AGTTTGACTC TTGAAGCCAT TTCAAACCAA
361 GGAACCATCC GCTTTGTTTG CAACTCATGG GTTTATAACA CTAAACTTTA CAAAAGCGTG
421 CGCATTTTCT TTGCCAACCA TACATATGTA CCTAGTGAGA CACCAGCACC ACTTGTGGAG
481 TACAGAGAAG AAGAATTGAA GAGTTTAAGA GGAAATGGAA CGGGAGAGCG CAAGGAATAT
541 GATAGGATCT ATGATTATGA TGTCTACAAT GATTTGGGCA ACCCAGATAA GAGTGAAAAG
601 TTAGCTCGTC CAGTTCTTGG AGGGTCTAGC ACCTTTCCCT ACCCTCGTAG GGGAAGAACT
661 GGTCGAGGTC CAACAGTTAC AGACCCGAAT ACTGAGAAAC AAGGCGAAGT ATTTTATGTT
721 CCAAGAGATG AAAATTTAGG TCACTTGAAG TCTAAGGATG CTCTTGAGAT TGGAACAAAA
781 TCTTTATCTC AAATTGTCCA GCCGGCGTTC GAATCTGCGT TCGATTTGAA ATCCACGCCA
841 ATTGAGTTTC ATAGCTTCCA AGATGTGCAT GACTTGTATG AAGGTGGAAT TAAGCTTCCT
901 AGAGATGTAA TTAGCACAAT TATCCCCTTA CCAGTGATCA AAGAACTCTA TCGTACCGAT
961 GGTCAACACA TCCTCAAGTT TCCACAACCT CACGTCGTTC AAGTGAGTCA GTCTGCATGG
1021 ATGACTGATG AAGAATTTGC TAGAGAGATG ATTGCTGGTG TAAATCCCTG CGTAATTCGT
1081 GGTCTTGAAG AGTTCCCTCC AAAAAGTAAT CTGGATCCTG CAATCTATGG TGATCAAAGC
1141 AGTAAGATAA CAGCAGATTC CCTTGATCTA GATGGGTACA CAATGGACGA GGCACTTGGT
1201 AGTCGAAGGT TATTTATGTT AGATTACCAT GATATCTTCA TGCCATATGT GAGGCAGATA
1261 AATCAGCTGA ATTCTGCCAA GACTTATGCG ACAAGGACTA TCCTTTTTTT GAGAGAAGAT
1321 GGAACTTTAA AGCCAGTGGC CATCGAATTA AGTTTGCCAC ATTCTGCTGG GGATCTGTCA
1381 GCTGCCGTCA GTCAAGTCGT CTTACCTGCT AAGGAAGGTG TTGAGAGCAC AATTTGGCTA
1441 CTAGCCAAAG CTTATGTCAT CGTAAATGAC TCTTGCTACC ATCAACTCAT GAGCCATTGG
1501 TTAAATACTC ATGCGGCGAT GGAGCCATTC GTCATAGCAA CACACCGACA TCTTAGCGTG
1561 CTTCACCCAA TTTACAAGCT TCTGACTCCT CACTATCGTA ACAACATGAA CATCAACGCA
1621 CTTGCCAGGC AATCTCTAAT TAATGCTAAT GGCATAATAG AGACAACCTT TTTGCCCTCA
1681 AAGTATTCTG TGGAGATGTC TTCGGCGGTT TATAAGAATT GGGTTTTCAC TGATCAAGCA
1741 CTACCTGCAG ATCTTATCAA GAGAGGAGTG GCAATTAAGG ATCCATCAAC CCCACATGGA
1801 GTTCGTCTTC TGATAGAGGA CTATCCTTAT GCCGCTGATG GACTGGAGAT ATGGGCTGCA
1861 ATTAAGACAT GGGTTCAAGA ATATGTGCCC TTGTACTATG CAAGAGATGA TGATGTCAAG
1921 AATGATTCTG AACTCCAACA TTGGTGGAAA GAAGCTGTAG AGAAAGGCCA TGGTGATTTG
1981 AAAGACAAGC CATGGTGGCC TAAGTTGCAG ACACTTGAAG ACCTTGTTGA AGTTTGCCTC
2041 ATTATCATAT GGATTGCTTC AGCTCTCCAT GCAGCCGTTA ATTTTGGTCA GTATCCTTAT
2101 GGAGGTTTGA TAATGAACCG CCCAACTGCT TCTAGAAGGT TGCTTCCTGA GAAAGGCACC
2161 CCAGAATATG AAGAAATGAT TAATAACCAT GAAAAGGCTT ATTTGAGAAC AATTACATCA
2221 AAGTTGCCGA CTCTCATTAG CCTTTCAGTG ATAGAGATCT TGTCGACACA TGCTTCTGAT
2281 GAGGTCTACC TTGGCCAGAG GGACAACCCA CATTGGACCT CTGATTCAAA AGCATTACAA
2341 GCCTTTCAAA AGTTTGGAAA CAAGCTCAAG GAAATTGAGG AAAAACTTGT GAGGAGGAAC
2401 AATGATCCGA GTCTGCAGGG CAATCGACTT GGCCCGGTTC AACTGCCATA CACTTTGCTT
2461 TATCCTAGCA GTGAGGAAGG GTTAACTTTT AGGGGAATTC CAAATAGCAT CTCTATCTAA
2521 ACTAGTCATG GGGCTCTAAC TCTCCTCTGG CCATGGCGAT ATCGGATCCG AATTCGAGCT
2581 CCGTCGACAA GCTTGCGGCC GCACTCGAGC ACCACCACCA CCACCACTGA GATCCGGCTG
2641 CTAACAAAGC CCGAAAGGAA GCTGAGTTGG CTGCTGCCAC CGCTGAGCAA TAACTAGCAT
2701 AACCCCTTGG GGCCTCTAAA CGGGTCTTGA GGGGTTTTTT GCTGAAAGGA GGAACTATAT
2761 CCGGATTGGC GAATGGGACG CGCCCTGTAG CGGCGCATTA AGCGCGGCGG GTGTGGTGGT
2821 TACGCGCAGC GTGACCGCTA CACTTGCCAG CGCCCTAGCG CCCGCTCCTT TCGCTTTCTT
2881 CCCTTCCTTT CTCGCCACGT TCGCCGGCTT TCCCCGTCAA GCTCTAAATC GGGGGCTCCC
2941 TTTAGGGTTC CGATTTAGTG CTTTACGGCA CCTCGACCCC AAAAAACTTG ATTAGGGTGA
3001 TGGTTCACGT AGTGGGCCAT CGCCCTGATA GACGGTTTTT CGCCCTTTGA CGTTGGAGTC
3061 CACGTTCTTT AATAGTGGAC TCTTGTTCCA AACTGGAACA ACACTCAACC CTATCTCGGT
3121 CTATTCTTTT GATTTATAAG GGATTTTGCC GATTTCGGCC TATTGGTTAA AAAATGAGCT
3181 GATTTAACAA AAATTTAACG CGAATTTTAA CAAAATATTA ACGCTTACAA TTTAGGTGGC
3241 ACTTTTCGGG GAAATGTGCG CGGAACCCCT ATTTGTTTAT TTTTCTAAAT ACATTCAAAT
3301 ATGTATCCGC TCATGAATTA ATTCTTAGAA AAACTCATCG AGCATCAAAT GAAACTGCAA
3361 TTTATTCATA TCAGGATTAT CAATACCATA TTTTTGAAAA AGCCGTTTCT GTAATGAAGG
3421 AGAAAACTCA CCGAGGCAGT TCCATAGGAT GGCAAGATCC TGGTATCGGT CTGCGATTCC
3481 GACTCGTCCA ACATCAATAC AACCTATTAA TTTCCCCTCG TCAAAAATAA GGTTATCAAG
3541 TGAGAAATCA CCATGAGTGA CGACTGAATC CGGTGAGAAT GGCAAAAGTT TATGCATTTC
3601 TTTCCAGACT TGTTCAACAG GCCAGCCATT ACGCTCGTCA TCAAAATCAC TCGCATCAAC
3661 CAAACCGTTA TTCATTCGTG ATTGCGCCTG AGCGAGACGA AATACGCGAT CGCTGTTAAA
3721 AGGACAATTA CAAACAGGAA TCGAATGCAA CCGGCGCAGG AACACTGCCA GCGCATCAAC
3781 AATATTTTCA CCTGAATCAG GATATTCTTC TAATACCTGG AATGCTGTTT TCCCGGGGAT
3841 CGCAGTGGTG AGTAACCATG CATCATCAGG AGTACGGATA AAATGCTTGA TGGTCGGAAG
3901 AGGCATAAAT TCCGTCAGCC AGTTTAGTCT GACCATCTCA TCTGTAACAT CATTGGCAAC
3961 GCTACCTTTG CCATGTTTCA GAAACAACTC TGGCGCATCG GGCTTCCCAT ACAATCGATA
4021 GATTGTCGCA CCTGATTGCC CGACATTATC GCGAGCCCAT TTATACCCAT ATAAATCAGC
4081 ATCCATGTTG GAATTTAATC GCGGCCTAGA GCAAGACGTT TCCCGTTGAA TATGGCTCAT
4141 AACACCCCTT GTATTACTGT TTATGTAAGC AGACAGTTTT ATTGTTCATG ACCAAAATCC
4201 CTTAACGTGA GTTTTCGTTC CACTGAGCGT CAGACCCCGT AGAAAAGATC AAAGGATCTT
4261 CTTGAGATCC TTTTTTTCTG CGCGTAATCT GCTGCTTGCA AACAAAAAAA CCACCGCTAC
4321 CAGCGGTGGT TTGTTTGCCG GATCAAGAGC TACCAACTCT TTTTCCGAAG GTAACTGGCT
4381 TCAGCAGAGC GCAGATACCA AATACTGTCC TTCTAGTGTA GCCGTAGTTA GGCCACCACT
4441 TCAAGAACTC TGTAGCACCG CCTACATACC TCGCTCTGCT AATCCTGTTA CCAGTGGCTG
4501 CTGCCAGTGG CGATAAGTCG TGTCTTACCG GGTTGGACTC AAGACGATAG TTACCGGATA
4561 AGGCGCAGCG GTCGGGCTGA ACGGGGGGTT CGTGCACACA GCCCAGCTTG GAGCGAACGA
4621 CCTACACCGA ACTGAGATAC CTACAGCGTG AGCTATGAGA AAGCGCCACG CTTCCCGAAG
4681 GGAGAAAGGC GGACAGGTAT CCGGTAAGCG GCAGGGTCGG AACAGGAGAG CGCACGAGGG
4741 AGCTTCCAGG GGGAAACGCC TGGTATCTTT ATAGTCCTGT CGGGTTTCGC CACCTCTGAC
4801 TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG GGGGGCGGAG CCTATGGAAA AACGCCAGCA
4861 ACGCGGCCTT TTTACGGTTC CTGGCCTTTT GCTGGCCTTT TGCTCACATG TTCTTTCCTG
4921 CGTTATCCCC TGATTCTGTG GATAACCGTA TTACCGCCTT TGAGTGAGCT GATACCGCTC
4981 GCCGCAGCCG AACGACCGAG CGCAGCGAGT CAGTGAGCGA GGAAGCGGAA GAGCGCCTGA
5041 TGCGGTATTT TCTCCTTACG CATCTGTGCG GTATTTCACA CCGCAATGGT GCACTCTCAG
5101 TACAATCTGC TCTGATGCCG CATAGTTAAG CCAGTATACA CTCCGCTATC GCTACGTGAC
5161 TGGGTCATGG CTGCGCCCCG ACACCCGCCA ACACCCGCTG ACGCGCCCTG ACGGGCTTGT
5221 CTGCTCCCGG CATCCGCTTA CAGACAAGCT GTGACCGTCT CCGGGAGCTG CATGTGTCAG
5281 AGGTTTTCAC CGTCATCACC GAAACGCGCG AGGCAGCTGC GGTAAAGCTC ATCAGCGTGG
5341 TCGTGAAGCG ATTCACAGAT GTCTGCCTGT TCATCCGCGT CCAGCTCGTT GAGTTTCTCC
5401 AGAAGCGTTA ATGTCTGGCT TCTGATAAAG CGGGCCATGT TAAGGGCGGT TTTTTCCTGT
5461 TTGGTCACTG ATGCCTCCGT GTAAGGGGGA TTTCTGTTCA TGGGGGTAAT GATACCGATG
5521 AAACGAGAGA GGATGCTCAC GATACGGGTT ACTGATGATG AACATGCCCG GTTACTGGAA
5581 CGTTGTGAGG GTAAACAACT GGCGGTATGG ATGCGGCGGG ACCAGAGAAA AATCACTCAG
5641 GGTCAATGCC AGCGCTTCGT TAATACAGAT GTAGGTGTTC CACAGGGTAG CCAGCAGCAT
5701 CCTGCGATGC AGATCCGGAA CATAATGGTG CAGGGCGCTG ACTTCCGCGT TTCCAGACTT
5761 TACGAAACAC GGAAACCGAA GACCATTCAT GTTGTTGCTC AGGTCGCAGA CGTTTTGCAG
5821 CAGCAGTCGC TTCACGTTCG CTCGCGTATC GGTGATTCAT TCTGCTAACC AGTAAGGCAA
5881 CCCCGCCAGC CTAGCCGGGT CCTCAACGAC AGGAGCACGA TCATGCGCAC CCGTGGGGCC
5941 GCCATGCCGG CGATAATGGC CTGCTTCTCG CCGAAACGTT TGGTGGCGGG ACCAGTGACG
6001 AAGGCTTGAG CGAGGGCGTG CAAGATTCCG AATACCGCAA GCGACAGGCC GATCATCGTC
6061 GCGCTCCAGC GAAAGCGGTC CTCGCCGAAA ATGACCCAGA GCGCTGCCGG CACCTGTCCT
6121 ACGAGTTGCA TGATAAAGAA GACAGTCATA AGTGCGGCGA CGATAGTCAT GCCCCGCGCC
6181 CACCGGAAGG AGCTGACTGG GTTGAAGGCT CTCAAGGGCA TCGGTCGAGA TCCCGGTGCC
6241 TAATGAGTGA GCTAACTTAC ATTAATTGCG TTGCGCTCAC TGCCCGCTTT CCAGTCGGGA
6301 AACCTGTCGT GCCAGCTGCA TTAATGAATC GGCCAACGCG CGGGGAGAGG CGGTTTGCGT
6361 ATTGGGCGCC AGGGTGGTTT TTCTTTTCAC CAGTGAGACG GGCAACAGCT GATTGCCCTT
6421 CACCGCCTGG CCCTGAGAGA GTTGCAGCAA GCGGTCCACG CTGGTTTGCC CCAGCAGGCG
6481 AAAATCCTGT TTGATGGTGG TTAACGGCGG GATATAACAT GAGCTGTCTT CGGTATCGTC
6541 GTATCCCACT ACCGAGATGT CCGCACCAAC GCGCAGCCCG GACTCGGTAA TGGCGCGCAT
6601 TGCGCCCAGC GCCATCTGAT CGTTGGCAAC CAGCATCGCA GTGGGAACGA TGCCCTCATT
6661 CAGCATTTGC ATGGTTTGTT GAAAACCGGA CATGGCACTC CAGTCGCCTT CCCGTTCCGC
6721 TATCGGCTGA ATTTGATTGC GAGTGAGATA TTTATGCCAG CCAGCCAGAC GCAGACGCGC
6781 CGAGACAGAA CTTAATGGGC CCGCTAACAG CGCGATTTGC TGGTGACCCA ATGCGACCAG
6841 ATGCTCCACG CCCAGTCGCG TACCGTCTTC ATGGGAGAAA ATAATACTGT TGATGGGTGT
6901 CTGGTCAGAG ACATCAAGAA ATAACGCCGG AACATTAGTG CAGGCAGCTT CCACAGCAAT
6961 GGCATCCTGG TCATCCAGCG GATAGTTAAT GATCAGCCCA CTGACGCGTT GCGCGAGAAG
7021 ATTGTGCACC GCCGCTTTAC AGGCTTCGAC GCCGCTTCGT TCTACCATCG ACACCACCAC
7081 GCTGGCACCC AGTTGATCGG CGCGAGATTT AATCGCCGCG ACAATTTGCG ACGGCGCGTG
7141 CAGGGCCAGA CTGGAGGTGG CAACGCCAAT CAGCAACGAC TGTTTGCCCG CCAGTTGTTG
7201 TGCCACGCGG TTGGGAATGT AATTCAGCTC CGCCATCGCC GCTTCCACTT TTTCCCGCGT
7261 TTTCGCAGAA ACGTGGCTGG CCTGGTTCAC CACGCGGGAA ACGGTCTGAT AAGAGACACC
7321 GGCATACTCT GCGACATCGT ATAACGTTAC TGGTTTCACA TTCACCACCC TGAATTGACT
7381 CTCTTCCGGG CGCTATCATG CCATACCGCG AAAGGTTTTG CGCCATTCGA TGGTGTCCGG
7441 GATCTCGACG CTCTCCCTTA TGCGACTCCT GCATTAGGAA GCAGCCCAGT AGTAGGTTGA
7501 GGCCGTTGAG CACCGCCGCC GCAAGGAATG GTGCATGCAA GGAGATGGCG CCCAACAGTC
7561 CCCCGGCCAC GGGGCCTGCC ACCATACCCA CGCCGAAACA AGCGCTCATG AGCCCGAAGT
7621 GGCGAGCCCG ATCTTCCCCA TCGGTGATGT CGGCGATATA GGCGCCAGCA ACCGCACCTG
7681 TGGCGCCGGT GATGCCGGCC ACGATGCGTC CGGCGTAGAG GATCGAGATC GATCTCGATC
7741 CCGCGAAATT AATACGACTC ACTATAGGGG AATTGTGAGC GGATAACAAT TCCCCTCTAG
7801 AAATAATTTT GTTTAACTTT AAGAAGGAGA TATACATATG CACCATCATC ATCATCATTC
7861 TTCTGGTCTG GTGCCACGCG GTTCTGGTAT GAAAGAAACC GCTGCTGCTA AATTCGAACG
7921 CCAGCACATG GACAGCCCAG ATCTGGGTAC CGGTGGTGGC TCCGGTATTG AGGGTCGC