Chemical synthesis, molecular cloning, and expression of the gene coding for the Trichosanthes trypsin inhibitor--a squash family inhibitor

The gene coding for a Trichosanthes trypsin inhibitor analog (Ala-6-TTI) in which methionine at position 6 was replaced by alanine was synthesized chemically. The synthetic gene was cloned into plasmid pWR590-1 and expressed in Escherichia coli as a fusion protein composed of beta-galactosidase fragment of 590 amino acid residues and (Ala-6)-TTI, with methionine as a connecting residue. After cyanogen bromide cleavage and reduction of the fusion protein, followed by refolding with trypsin-Sepharose 4B as a matrix and affinity chromatography on the immobilized enzyme, the fully active (Ala-6) -TTI was obtained. The trypsin inhibitory activity and amino acid composition of the recombinant (ALA-6)-TTI were consistent with those of the natural one. The (ALA-6)-TTI gene was also cloned into the secretion expression vector, pVT102U/alpha, in Saccharomyces cerevisiae. In order to make the reading frame of the gene compatible with the vector, a nucleotide was inserted into the (ALA-6)-TTI gene via site-directed mutagenesis. The secreted (ALA-6)-TTI was purified and found to be correctly processed at the junction between the alpha-factor leader peptide and (ALA-6)-TTI downstream. Of the two expression systems, the latter is more advantageous in the high yield (> 2 mg/liter), easy purification and needlessness of disulfide refolding.