The coding region of the Ti a allelic form of the soybean (Kunitz) trypsin inhibitor (SBTI) gene ( KTi3 ) has been introduced, as a transcriptional fusion with the CaMV 35S promoter, into white clover ( Trifolium repens L.). Southern analysis of DNA extracted from primary transformants confirmed that an intact copy (or copies) of the gene is integrated into the white clover genome. Gel filtration column chromatography of transgenic leaf tissue revealed a higher molecular mass (ca. 22 kDa) trypsin inhibitory fraction, and a lower molecular mass (ca. 15 kDa) trypsin inhibitory fraction. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using an anti-SBTI monoclonal antibody (MAb) determined that SBTI occurred in the higher molecular mass fraction as a protein of ca. 22 kDa. Gel filtration column chromatography revealed that the lower molecular mass trypsin inhibitory fraction also occurred in non-transgenic leaf tissue. Further purification of the SBTI inhibitory fraction using reverse-phase column chromatography revealed two major trypsin inhibitory peaks, only one of which was recognised by the SBTI MAb. N-terminal sequencing confirmed that this antibody-recognised inhibitor is the KTi3 gene product, and the protein is processed by the removal of the N-terminal leader sequence. N-terminal sequencing of the major protein in the second reverse-phase trypsin inhibitory fraction produced no matches to any inhibitors in the database. Further purification of the lower molecular mass trypsin inhibitory protein using reverse-phase chromatography from either transgenic or non-transgenic leaf tissue revealed a single trypsin inhibitory fraction (designated WCTI-1). N-terminal sequencing produced a match with non-specific lipid transfer proteins from higher plants. Kinetic analysis of WCTI-1 determined an equilibrium constant ( K I value) of 1.2 × 10 −8 M for trypsin, and showed that the inhibitor was effective against trypsin-like activity of black field cricket ( Teleogryllus commodus ) and grass grub ( Costelytra zealandica ), but ineffective against trypsin-like activity from light brown apple moth ( Epiphyas postvittana ) and porina ( Wiseana copularis ).
