Expression of Fragaria ananassa Osmotin-like Protein(FaOLP2) in E. coli:Purification and Antifungal Activity

查看全文 作　　者：Bai [1,2]LI;Sufang [1]LI;Huandi [1]LI;Junwei [1]SUN;Keqin [1]ZOU 机构地区：[1]College of Life Sciences,Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine,China Jiliang University;[2]Jiaxing Academy of Agricultural Sciences 出　　处：《Agricultural Biotechnology》索引2013年第2卷第6期,共5页 基　　金：Supported by Zhejiang Province Natural Science Foundation(Y307591,Y3110288);Key Scientific and Technological Innovation Team Program of Zhejiang Province(2010R50028) 摘　　要：[Objective]The FaOLP2(Fragaria ananassa osmotin-like protein)is a functional homolog of PR5-like protein.This study was undertaken to produce recombinant FaOLP2 and to identify its antifungal activity.[Method]The ORF of FaOLP2(accession number DQ325524)was cloned into pET22b vector to construct the pET22b-FaOLP2 plasmid.The recombinant mature FaOLP2 was expressed in E.coli Rosetta-gami B(DE3)by inducing with 1 mmol/L IPTG and found exclusively in insoluble inclusion bodies.As FaOLP2 requires the correct formation of eight disulfide bonds,but there were no obvious effect to correctly form these by expression at different temperatures and high osmotic pressure(supplemented Betaineand and D-Sorbitol),we used an in vitro method to refold E.coli expressed FaOLP2 by gradually elution using reduced:oxidized gluthatione redox buffer,followed by 8 mol/L urea solubilized His6-tagged mature FaOLP2 protein,which was affinity-purified by an immobilized-metal(Ni2+)affinity chromatography(IMAC)column.[Result]This method generated biologically active conformations of the recombinant mature FaOLP2 that displayed antifungal activity against Ustilaginoides virens,a plant pathogenic fungus,which causes rice false smut.[Conclusion]This study laid the foundation for further biotechnological application of the novel protein. 关 键 词：大肠杆菌表达 亲和纯化 蛋白质 逆渗透 抗菌活性 抗真菌活性 草莓 生产重组