Expression, purification and activity identification of oxysterol-binding protein from Phytophthora sojae in Escherichia coli

Oxathiapiprolin is a fungicide with high actvity currently used in the field to prevent and treat plant pathogen oomycete disease, and its target has been supposed to be an oxysterol-binding protein (OSBP). OSBP and OSBP-related proteins (ORPs) as lipid transfer proteins are conserved in various species. Although the three-dimensional structure of this protein in other species has been elucidated, there have been no reports on its heterologous expression and purification in plant pathogenic oocytes, which limits the structural pharmacology research of this protein and the development of new targeted fungicides. This article aims to establish a system including in vitro expression, purification, and activity identification for the OSBP from Phytophthora sojae (Ps),Psosbp. In this study, the OSBP related domain (ORD) conserved with the human OSBP sequence was selected to construct a plasmid suitable for expression in Escherichia coli and the protein expression was induced by isopropyl-β-D-thiogalactoside (IPTG). The target protein was purified by affinity chromatography and gel filtration chromatography, respectively. Then protein was identified using SDS-PAGE and Western blot. Ultimately, the structural integrity and activity of protein were detected by Tycho™ NT.6 and microscale thermophoresis (MST), respectively. The result showed that amino acids from amino acid 600th to 967th of Psosbp protein sequence, namely Psosbp (600-967), were selected as ORD domain of Psosbp and used to construct the expression plasmid pET28a-MBP-TEV-Psosbp (600-967)-His6. IPTG could induce the expression of the target protein and the protein was soluble. The protein was affinity purified using Ni-NTA agarose resin, and a highly purified recombinant proein MBP-TEV-Psosbp (600-967)-His6 was further seperated by molecular exclusion chromatography. The result of Tycho™ NT.6 showed that the recombinant protein had a complete spatial structure, and MST analysis demonstrated that oxathiapiprolin could bind to the recombinant protein, indicating that the purified OSBP from P. sojae had biologic activity. In summary, this article established a system including heterologous expression, purification, and activity identification for OSBP from plant pathogenic oomycete, and directly confirmed that the inhibitor oxathiapiprolin could bind to OSBP.