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 (greater than 2 mg/liter), easy purification and needlessness of disulfide refolding.

Toluene mineralization and growth potential of Pseudomonas putida PaW164 under toluene-limiting conditions.

Toluene-induced cells of Pseudomonas putida PaW164 (pWWO-164) were monitored for growth potential, maintaining the TOL plasmid, and potential toluene mineralization activity in toluene-amended and nonamended soil. A follow-up study was done in a carbon-free mineral salts solution to obtain further information on physiological changes that occur during starvation. These studies showed that there was a larger decline in colony forming units (CFUs) recovered on a toluate- or benzoate-defined mineral salts medium than on a complex agar medium, a greater percent decrease of CFU than of potential mineralization activity, no decrease in direct counts, and no loss of the TOL plasmid during starvation. Toluene-induced cells also showed an increasing lag time and a decreasing potential for mineralization of (14)C-toluene with starvation. In contrast, the lag time for mineralization of glucose was longest at the onset of starvation and reached a minimum by 3 days; thereafter, the potential for glucose mineralization remained high.