集胞藻PCC6803中脂肪酸激活酶Slr1609互作蛋白的鉴定  

Identification and characterization of partner proteins interacting with fatty acid activation enzyme Slr1609 in Synechocystis sp. PCC 6803

作　　者：徐乐[1] 巫琴[1] 晋虎[1] 陈磊[1] 张卫文[1] 

机构地区：[1]天津大学化工学院合成微生物学实验室,天津300072

出　　处：《生物工程学报》2015年第8期1194-1202,共9页Chinese Journal of Biotechnology

基　　金：国家重点基础研究发展计划(973计划)(Nos.2012CB721101;2011CBA00803)资助~~

摘　　要：集胞藻中slr1609是编码脂肪酸激活酶的基因,对与其相关的重要功能伴侣蛋白进行研究,可以完善对脂肪酸合成模块的认识,为进一步通过合成生物学技术改造蓝细菌提供理论支持。本研究在集胞藻PCC 6803中建立了蛋白质复合体分析及鉴定技术:利用氯霉素抗性基因筛选,构建带有3×FLAG标签的Slr1609突变株,通过RT-PCR优化重组蛋白表达条件;同时对突变株进行了Western blotting鉴定,以及利用Native-PAGE验证了蛋白质复合体的存在。最后,LC-MS/MS质谱鉴定获得了Slr1609蛋白复合体中的可能伴侣蛋白。To understand molecular modules related to polyunsaturated fatty acids （PUFA） synthesis and eventuallyproduce PUFA at high efficiency, we developed a protein complex analysis technology in Synechocystis sp. PCC 6803, and applied it to identify possible partner proteins interacting with the key enzymes that catalyze PUFA biosynthesis. We first constructed a recombinant expression of protein of slr1609 encoding the fatty acid activation enzyme, by fusing 3×FLAG tag with the target protein. Then we verified its expression by Western blotting targeting 3×FLAG tag. To maximize purification of Slr1609 protein complex, we optimized the protein expression conditions of Slr1609 in Synechocystis in a 5 L fermenter by monitoring its gene expression using RT-qPCR. The purification of the Slr1609 protein complexes was demonstrated by a Native-PAGE analysis. Finally, LC-MS/MS proteomic analysis allowed identification of the possible partner proteins interacting with Sir 1609.

关 键 词：slr1609 脂肪酸 蛋白质复合体 蓝细菌 

分 类 号：Q946[生物学—植物学]