人源脂肪酸合酶在酿酒酵母中的表达纯化和结构的初步分析

[背景] 聚酮类化合物在医药领域有重要的应用，相关药物研发依赖聚酮合酶多变的结构认知，人源脂肪酸合酶的组成结构和催化机制与聚酮合酶相近，研究人源脂肪酸合酶结构可为聚酮合酶的研究奠定基础。[目的] 在酿酒酵母中表达纯化人源脂肪酸合酶蛋白，确定合适的体外纯化条件。[方法] 以酿酒酵母BJ5464为表达载体，构建带有His和Strep双亲和层析标签的重组质粒，诱导表达蛋白后用亲和层析方法获取目标蛋白，并结合凝胶电泳和快速蛋白质液相层析技术，确定合适的蛋白纯化条件。[结果] 成功构建重组表达质粒pxw55-hfas-cSHII， 并在体外纯化得到合适浓度和纯度的人源脂肪酸合酶蛋白，筛选不同缓冲液条件并结合电子显微镜观察结果反馈，确定合适的蛋白体外纯化体系。[结论] 蛋白电镜结构分析需要有高纯度、合适浓度并且形成正确构象的蛋白样品，而人源脂肪酸合酶蛋白纯化体系的建立和纯化条件的确定为其电镜结构分析提供了良好的样品，为人源脂肪酸合酶的结构解析及结构相似但更为复杂的聚酮合酶蛋白解析奠定了良好基础。

Expression, purification and preliminary structural analysis of human fatty acid synthase in Saccharomyces cerevisiae

[Background] Polyketides have important applications in the pharmaceutical field, and the development of related drugs relies on the variable structural knowledge of polyketide synthases. The structural and catalytic mechanism of human fatty acid synthase are similar to those of polyketide synthase, and the study of human fatty acid synthase structure can lay the foundation for the study of polyketide synthase. [Objective] We worked on the expression of purified human fatty acid synthase protein in Saccharomyces cerevisiae and determined suitable in vitro purification conditions. [Methods] The recombinant plasmid with His and Strep dual affinity chromatography tags was constructed using Saccharomyces cerevisiae BJ5464 as the expression vector, and the target protein was purified by affinity chromatography after the protein overexpression, and the suitable protein purification conditions were determined by gel electrophoresis results. [Results] The recombinant expression plasmid pxw55-hfas-cSHII was successfully constructed and the human fatty acid synthase was purified in vitro. Preliminary screening under different buffer conditions was tried and the appropriate protein purification system was determined by combining with the feedback of electron microscope observation results. [Conclusion] Structural analysis by electron microscopy requires high protein purity, suitable concentration and correct conformation of protein samples. The construction of the purification system and the determination of the purification conditions of human fatty acid synthase provided a good sample for the analysis of the structure of human fatty acid synthase, and pave the way for the analysis of the similar but more complicated polyketide synthase.