Purification and characterization of a trypsin inhibitor from Senna tora active against midgut protease of podborer

Abstract

Proteinaceous protease inhibitors have potential application in medicines, agriculture and biotechnology. Present study was undertaken to purify and characterize a proteinaceous protease inhibitor from a medicinal plant, Senna tora syn. Cassia tora. The inhibitor was purified by ammonium sulphate precipitation, anion exchange (Q-sepharose), affinity (trypsin-sepharose) and molecular exclusion (sephadex G-75) chromatography. Zymography and denaturing polyacrylamide gel electrophoresis revealed a single band of ∼20kDa trypsin inhibitor. Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Matrix-assisted laser desorption ionization (MALDI) analyses revealed the presence of 19,725Da (pI 4.60) and ∼19,900Da (pI 4.57) isoform proteins in purified inhibitor. Protein identification by MALDI-peptide mass fingerprinting did not reveal high MASCOT (Matrix science) scores matching with previously known inhibitors. N-terminal amino acid sequence suggested this protein as a previously unreported inhibitor. Its dissociation constant (0.23×10−9M) was indicative of a high affinity trypsin inhibitor. The inhibitor was stable over a broad range of pH (4–10) and temperature (30–60°C). The purified inhibitor effectively inhibited total protease and trypsin-like activities of podborer (Helicoverpa armigera) midgut preparation. Hence, the inhibitor and its gene(s) can find application in combating against pest and protease dependent pathogens.