海洋放线菌S.areniocola CNS-205腺苷化结构域基因的克隆、表达和纯化  

Cloning,expression and purification of a gene of adenylation domain from marine actinomycete S.arenicola CNS-205

作　　者：陆胜利[1] 祁超[2] 

机构地区：[1]安庆医药高等专科学校药学系,安徽安庆246052 [2]华中师范大学生命科学学院,武汉430079

出　　处：《华中师范大学学报（自然科学版）》2014年第6期876-884,共9页Journal of Central China Normal University：Natural Sciences

基　　金：国家自然科学基金项目(20772040);教育部中央高校自主项目(CCNU14F01006)

摘　　要：海洋放线菌S.arenicola CNS-205是首次报道的专属海洋性放线菌属Salinispora的代表菌株之一.选取海洋放线菌S.arenicola CNS-205非核糖体多肽合成酶氨基酸腺苷化结构域基因sare 0357,对其进行生物信息学分析后进行原核重组质粒的构建和表达及蛋白纯化.分别构建了pET-28a-sare 0357和pGEX-KG-sare 0357重组质粒,并转化E.coli BL21(DE3)进行不同诱导条件的诱导表达,sare 0357基因均以包涵体的形式表达.收集Sare0357重组蛋白包涵体,选取6mol/L的盐酸胍对其进行变性并将变性后的重组蛋白进行镍柱纯化.得到了可溶性的Sare0357融合蛋白.Salinispora arenicola CN8-205 was a firstly-isolated obligate marine actinomycete. Several bioactive metabolites, including NRP compounds, have been isolated from this strain. Existence of a high number of secondary metabolites biosynthetic clusters on its genome revealed its strong potential to accumulate more natural products. Nonribosomal peptide synthetases （NRSs） are critical enzymes for the biosynthesis of nonribosomal peptide （NRP） compounds,a large class of biologically active natural products. Amino acid adenylation domains （A domains） are essential components within NRPSs and recognize specifically amino acid buiding blocks to be incorporated during the biosynthesis of NRPSs. a gene sare0357 annotated as ＂amino acid adenylation domain＂ located on the genome of Salinispora arenicola CNS-205, was chined and characterized. The recombinant target protein Sate0357 was expressed efficiedntly but as inclusion bodies in a prokaryotie system. After denaturation and renaturation and purification, soluble protein was obtained.

关 键 词：海洋放线菌 非核糖体多肽合成酶 腺苷化结构域 

分 类 号：Q812[生物学—生物工程]