SYNTHESIS, CLONING AND EXPRESSION IN ESCHERICHIA-COLI OF A GENE CODING FOR THE MET8-]LEU-CMTI-I - A REPRESENTATIVE OF THE SQUASH INHIBITORSOF SERINE PROTEINASES

A chemically synthesized gene coding for a Cucurbita maxima trypsin in hibitor modified at position P'3 (Met8 --> Leu CMTI I), i.e. at the th ird position downstream of the reactive site bond (Arg5-Ile), was clon ed into a derivative of the plasmid pAED4 that utilizes a T7 expressio n system, The gene was expressed in Escherichia coli as a fusion prote in that accumulates in inclusion bodies, After reduction and CNBr clea vage of the fusion protein followed by oxidative refolding and reverse -phase HPLC, about 5 mg of pure protein was obtained per 1 of cell cul ture, Association constants of recombinant Leu-8-CMTI I with bovine be ta-trypsin and human cathepsin G are the same, within experimental err or, as for CMTI I isolated from a natural source.