Molecular Cloning, Prokaryotic Expression And Characterization Of A Novel Trypsin Inhibitor In The Frog, Lepidobatrachus Laevis

Amphibian is a biological treasure with great potentials as well as important natural resources with ecological, research, economic and cultural value. Amphibian is a biological treasure with great potentials. To be important natural resources, the ecological, academic, economic and cultural values of this kind of organism become more and more important. Varieties of bio-active substances in amphibian skins which play an important role in defense against invading factors have attracted the researchers all over the world. According to the specific active site of residues, protease is divided into four categories:aspartic proteases, metalloproteinases, serine proteases and cysteine proteases. Accordingly, the protease inhibitors are divided into four kinds. By action mechanism, serine protease inhibitors can be divided into three types:canonical, non-canonical, serpin(serine protease inhibitors).This research object is Lepidobatrachus laevis, which is the unique frog in South America. We constructed it's skin cDNA library and selected it by randomly sequencing. We got two candidate genes:Y1-2, Y3-34. By amino acid sequence comparison, Y1-2 has 44% homology with trypsin inhibitor (Aedes aegypti). Y3-34 has 53% homology with hemagglutinin (Xenopus tropicalis). The position and number of their cysteines are consistent with the similar proteins.By pET expression system of E. coli, Y1-2 with trypsin inhibitor activity, is successfully expressed The purified Y1-2 is 6160.0273 daltons and consistent with the prediction. The Ki for Y1-2 is about 16.5178μM, which is determined by Dixon plot.