Synthesis, cloning and expression in Escherichia coli of a gene coding for the Met8->Leu CMTI I - A representative of the squash inhibitors of serine proteinases

A chemically synthesized gene coding for a Cucurbita maxima trypsin inhibitor modified at position P'3 (Met8 --> Leu CMTI I), i.e. at the third position downstream of the reactive site bond (Arg5-Ile), was cloned into a derivative of the plasmid pAED4 that utilizes a T7 expression system, The gene was expressed in Escherichia coli as a fusion protein that accumulates in inclusion bodies, After reduction and CNBr cleavage of the fusion protein followed by oxidative refolding and reverse-phase HPLC, about 5 mg of pure protein was obtained per 1 of cell culture, Association constants of recombinant Leu-8-CMTI I with bovine beta-trypsin and human cathepsin G are the same, within experimental error, as for CMTI I isolated from a natural source.