Prokaryotic expression, purification, physicochemical properties and antifungal activity analysis of phloem protein PP2-A1 from cucumber

Abstract

Phloem protein 2 (PP2) is a protein having lectin properties that can be isolated from the phloem sap. Based on our previous proteomic study of phloem sap of Cucumis sativus, it was found that the expression of PP2 A1-like was significantly up-regulated under salt stress, which may be a molecular mechanism of plant adaptation to stress. This paper carried out the expression and purification of the CsPP2-A1 gene in E. coli for further characteristic analysis. The results demonstrated that the CsPP2-A1 in shake flask cultures was mainly expressed in the soluble form at 15 °C or in inclusion bodies at 37 °C. Secondly, Ni-IDA affinity chromatography and SDS-PAGE were employed to yield highly purified CsPP2-A1 protein. The purified CsPP2-A1 was then subjected to Western blot and MALDI-TOF-MS analysis for protein identification. The biological activity analysis results showed that CsPP2-A1 had hemagglutinating activities to rabbit erythrocytes, and Chitotetraose may be the specific inhibitory sugar of CsPP2-A1. The optimal hemagglutination activity of CsPP2-A1 protein was achieved between pH 5–9, and between 20 and 60 °C. Moreover, CsPP2-A1 had significant inhibitory effects on Botrytis cinerea and Phytophthora infestans, and the inhibitory effect on B. cinerea was better than that on P. infestans.