Chemical synthesis, molecular cloning and expression of gene coding for a Bowman-Birk-type proteinase inhibitor.
A gene coding for a Bowman-Birk-type proteinase inhibitor was synthesized chemically, cloned and expressed in Escherichia coli as a fusion protein with a beta-galactosidase fragment. The corresponding mutant inhibitor, carrying a P1 = Arg16 instead of Lys and an Ile27 instead of Met was obtained after cyanogen bromide cleavage, refolding and affinity chromatography on trypsin-Sepharose. Dissociation constants of complexes with trypsin of this mutant and wild-type Bowman-Birk inhibitor are identical within experimental error. This is explained by differential patterns of hydrogen bonds between side-chains of Arg or Lys in proteinase inhibitors and the primary specificity pocket of trypsin.
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D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003488:Cyanogen Bromide; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D009154:Mutation; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014170:Transformation, Genetic; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000235:genetics*; D014361:Trypsin Inhibitors / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D014361:Trypsin Inhibitors; D003488:Cyanogen Bromide