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

Abstract:

[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.