Molecular cloning and characterization of novel cysteine protease inhibitors from Calotropis procera R. Br.

Abstract

The cysteine protease is an important enzyme in industrial, physiological, and therapeutic uses. This enzyme was found out to be a significant role in the autolysis in foods and the proliferation of tumor cells. Therefore, it needs to be inhibited during food processing and can be the target enzyme for drugs. For these reasons, effective cysteine protease inhibitors from natural sources have been studied for decades. Calotropis procera R. Br. was selected as an inhibitor source. It is a medicinal and edible tropical plant. In the preliminary study, its cDNA sequence and putative cysteine proteases were revealed by RNA sequencing. Propeptides of cysteine proteases from Calotropis procera R. Br. were considered to be efficient inhibitors against cathepsin L. In this study, the molecular cloning, expression, and characterization of several candidate cysteine protease inhibitors were described, and the cause that occur the inhibitory activity difference was presumed by bioinformatic analysis. Firstly, eight kinds of cysteine protease propeptides were selected as candidate inhibitors by sequence analysis with cathepsin L. They were cloned and expressed in Escherichia coli BL21(DE3). Three inhibitors (SnuCalCpI02, SnuCalCpI03, and SnuCalCpI15) were overexpressed in soluble form, and two inhibitors (SnuCalCpI12 and SnuCalCpI16) were overexpressed in mostly insoluble form. The others (SnuCalCpI08, SnuCalCpI14, and SnuCalCpI17) were not expressed. Then, the characterization of five expressed inhibitors was performed. Only two (SnuCalCpI03 and SnuCalCpI15) exhibited inhibitory activity against papain, and their inhibitory activity against human cathepsin L was investigated. The half maximal inhibitory concentrations (IC50) of SnuCalCpI03 and SnuCalCpI15 were 18.58 nM and 17.50 nM, respectively. They acted as the competitive inhibitors. The inhibitor proteins were stable at a high temperature and a wide range of pH except pI point. Lastly, to analyze the cause of the different inhibitory activity of inhibitors, bioinformatic analysis was carried out. Consequently, it is presumed that the inhibitory activity can be different by the tendency that inhibitors interact with the enzyme. Novel cysteine protease inhibitors from Calotropis procera R. Br. are expected to be used as efficient anti-tumor drugs or food stabilizers.