Chemical synthesis, molecular cloning and expression of gene coding for a Bowman-Birk-type proteinase inhibitor

Eur J Biochem. 1987 Jul 1;166(1):151-6. doi: 10.1111/j.1432-1033.1987.tb13495.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Chromatography, Affinity
  • Cloning, Molecular*
  • Cyanogen Bromide
  • Escherichia coli / genetics
  • Genes*
  • Mutation
  • Plasmids
  • Recombinant Fusion Proteins / genetics
  • Transformation, Genetic
  • Trypsin Inhibitor, Bowman-Birk Soybean / genetics*
  • Trypsin Inhibitors / genetics*

Substances

  • Recombinant Fusion Proteins
  • Trypsin Inhibitor, Bowman-Birk Soybean
  • Trypsin Inhibitors
  • Cyanogen Bromide