Chemical synthesis, molecular cloning, overexpression, and site-directed mutagenesis of the gene coding for pumpkin (Curcubita maxima) trypsin inhibitor CMTI-V.

The gene encoding for a pumpkin (Curcubita maxima) trypsin inhibitor CMTI-V was synthesized chemically. The synthetic gene was prepared from four overlapping oligonucleotides by overlapping extension. The synthetic gene was amplified by polymerase chain reaction, cloned into a T7 expression vector and expressed in Escherichia coli as a fusion protein. The clone, namely 70-1, encoded a fusion protein containing 7 amino acid residues of the N-terminus of the bacterial protein rho 10 and the entire 68 residues of CMTI-V. The wild-type fusion protein constituted approximately 15% of the total bacterial protein mass and was purified to homogeneity in a single step by antibody affinity chromatography. The wild-type fusion protein possesses inhibitory activity toward trypsin and beta-Factor XIIa, but to a lesser extent when compared to the natural CMTI-V. A mutant, T43A, in which threonine at position 43 (P2 position) was replaced by alanine, was constructed. This mutant showed considerably lower specific inhibitory activity toward both trypsin and beta-Factor XIIa.