集胞藻PCC 6803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

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。

Abstract:

To understand molecular modules related to polyunsaturated fatty acids (PUFA) synthesis and eventually produce 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 Slr1609.