BPI_(23)-haFGF融合蛋白在毕赤酵母中表达条件的优化

Optimization of Condition for Expression of BPI_(23)-haFGF Fusion Protein in Pichia pastoris

目的:通过优化毕赤酵母的表达参数,为提高BPI23-haFGF融合蛋白在毕赤酵母中的表达量创造条件。方法:采用小型摇瓶培养系统,对重组毕赤酵母菌X-33/pPICZαA-BPI23-haFGF在含有蛋白酶抑制剂PMSF以及不同诱导时间、培养温度、甲醇浓度及培养液pH值条件下进行表达,将表达上清进行SDS-PAGE电泳,并用Quantity one Volume Rect Tool和Dentity tool进行分析。结果:在诱导培养基中加入0.5mmol/L PMSF后,BPI23-haFGF融合蛋白降解得到缓解,诱导表达96h表达量达到高峰;在培养温度28℃,甲醇浓度为1.0%,培养液pH 6.0时,BPI23-haFGF表达量最高,含量比优化前提高了91.6%。结论:该研究为BPI23-haFGF融合蛋白后期的大规模发酵及深入研究其生物学功能奠定了基础。

Objective：To improve the expression of BPI23 - haFGF fusion protein by optimizing the parameters in Pichia pastoris. Method： The recombinant X - 33/pPICZαA - BPI23- haFGF was expressed at different induction time, cuhure - inducing temperature, methanol concentration, and the medium pH in a small flask culture system. Then the product of expression was analyzed by SDS - PAGE and Quan- tity one Volume Rect Tool and Dentity tool, in order to obtain better condition for expression. Result ：The degradation of BPI23 -haFGF is decreased in the induction medium with 0. 5 mmol/L PMSF; the expression of BPI23- - haFGF reaches a peak at 96h; the expression of tar- get protein are highest when the cuht, re - inducing temperature, is 28 ℃, the methanol concentration is I. 0%, the medium pH is 6. 0, Conclusion ：The study has laid a foundation for the large - scale preparation of BPI23- - haFGF to meet the needs of its biological research.