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 β-trypsin and human cathepsin G are the same, within experimental error, as for CMTI I isolated from a natural source.
