Expression, Purification And Biological Activity Of Recombinant Mung Bean Trypsin Inhibitor Fragments

Protease inhibitors are widely distributed in the animal and plant. It shares extensive physiological roles. Protease inhibitors not only inhibit the activities of enzymes, but also have significant anti-cancer function by suppressing cell invasion and promoting cell apoptosis. In accordance with these findings, Bowman-Birk inhibitor (BBI) has been extensively studied due to their potential anti-cancer activities. As an effective anti-cancer factor, BBI has many advantages, for instance:Its inhibition on tumor is irreversible, and BBI can effectively inhibit tumor cell growth and migration at low concentrations. The application range thereby is much larger than other anticancer agents. However, their anticancer mechanisms are less known yet, so the study about the mechanism of trypsin inhibitor on tumor cells is very important.Mung bean trypsin inhibitor, one of the most studied Bowman-Birk protease inhibitors, contains two active sites (Lys and Arg) and has a molecular weight of8kDa. We have known that there are more studies on its crystal structures and Physical and chemical properties rather than the biological activities. In this thesis, we focus on the effects of Lys active fragment on cancer cells and the possible mechanisms.In this study, a Lys active fragment of mung bean trypsin inhibitor, LysGP33, was successfully expressed in Escherichia coli as a glutathione-S-transferase fusion protein. The recombinant product was obtained with a high yield, and exhibited potent inhibitory activity on cancer cells. The changes of cell proliferation treated with GST-LysGP33were observed by MTT assay. Migration was examined by wound healing assay. We determined the effects of GST-LysGP33on levels of proteins associated with migration and invasion in SW480, HT-29, DLD1. The studies have shown that GST-LysGP33inhibited MMP-9expression which was activated through Wnt/β-catenin Signaling pathway. Recombinant mung bean trypsin inhibiotor fragment has some prospects in anti-tumor migration and invasion.