Study Of Recombinant Buckwheat Trypsin Inhibitor Active Site And Its Mutants

Protease inhibitors (PIs) are widely present in animals, plants and microorganisms. They play a significant role in lots of biological processes. Formation of stable complexes between inhibitors and enzymes is essential for life cycle. So some researchers have focus their aim on protease inhibitors. Up to now, there are many kinds of inhibitors have been isolated and characterized, fractional has become an excellent model in new drug design and development.Plants are the best source of protease inhibitors. The plant serine protease inhibitors are ubiquitious and widely studied. According to the sturcture homology, position of reactive sites and disulphide bridges, plant serine inhibitors can be divided into Kunitz, Bowman-Birk, Cucurbit, Potato I, Potato II and other families. Most studies of inhibitors are focus on Bowman-Birk, Kunitz and Potato I family at present.Buckwheat is a traditional farm crops of China and now widely cultivated around the world. Even though it is not a mainly crop, it remains essential for food supply in the temperate and hilly regions in southwest of China. As a kind of health food, buckwheat has been received more and more people's favor. It has been proved that some kinds of inhibitor from buckwheat could prevent the proliferation of human tumor cells. Previously, we had isolated and purified buckwheat trypsin inhibitor (BTI) from buckwheat seeds, and a recombinant BTI (rBTI) also was expressed in Escherchia coli M15 [pREP4]. In vitro, rBTI can inhibit the proliferation to several types of human tumor cells, while it has shows less toxicity against normal human cells. Thus, more research is necessary to fully characterize the structure-functional relationship of rBTI.In this study, site-directed mutagenesis was used to analyze structure-function relationship in rBTI. Using sequence comparison to other plant trypsin inhibitors, two mutants (R45A-aBTI, R45F-fBTI) were generated and assayed for trypsin inhibitor activity in order to determine the reactive site of rBTI. In addition, the antiproliferation effect of two mutants were researched, and compared with wild rBTI. In conclusion, knowledge about the active site is useful for further clarifying the physiological mechanism of rBTI and other protease inhibitors. New inhibitory activities of aBTI and fBTI are potentially useful in the fields of health and medicine. This study provides a foundation for further research, and we believe the results are valuable for understanding the structure-function relationship in protease inhibitors.