人血小板衍生生长因子-BB在酿酒酵母中的表达及纯化工艺的建立

Development of A Procedure for Expression of Human Platelet-Derived Growth Factor-BB in Saccharomyces cerevisiae and Purification of Expressed Product

目的建立重组人血小板衍生生长因子-BB(hPDGF-BB)在酿酒酵母中的表达及纯化工艺。方法在5L发酵罐中,探讨接种量、溶氧、诱导温度和诱导pH值对菌体密度及目的蛋白表达量的影响,并通过离子交换层析和凝胶过滤层析对目的蛋白进行纯化。结果重组hPDGF-BB工程菌在5L发酵罐分批补料培养中,经84h发酵,细胞A600值可达65.1,目的蛋白表达量占26%。纯化后的蛋白纯度可达95%,产量为15.4mg/L,收率为21.9%。结论已建立了周期短、产率高的hPDGF-BB发酵及纯化工艺,为其大规模生产奠定了基础。

Objective To develop a procedure for expression of human platelet-derived growth facto（rhPDGF）-BB in Saccha-romyces cerevisiae and purification of expressed product. Methods The influence of inoculum size,dissolved oxygen （DO）as well as temperature and pH values for induction on bacterial density of recombinant S. cerevisiae and expression level of target protein in 5 L fermentor were explored,based on which the expressed target protein was purified by ion exchange and gel filtration chromatography. Results Eighty-four hours after culture of recombinant S. cerevisiae carrying hPDGF-BB gene in 5 L fermentor by fed batch,the bacterial density （A600）reached 65. 1,and the expressed product contained 26% of total somatic protein and reached a purity of 95% after purification. The yield and recovery rate of target protein were 15. 4 mg / L and 21. 9% respectively. Conclusion A time-saving and effective procedure for fermentation of recombinant S. cerevisiae carrying hPDGF-BB gene and purification of expressed product was developed,which laid a foundation of large-scale production of hPDGF-BB.