Chemical Synthesis, Molecular Cloning, and Expression of the Gene Coding for the Trichosanthes Trypsin Inhibitor—a Squash Family Inhibitor1

Publisher Website
Google Scholar
Abstract
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-l and expressed in Escherichia coli as a fusion protein composed of β-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/α, 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 α-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.

This publication has 0 references indexed in Scilit: