【摘要】 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。更多还原

【Abstract】 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,culture-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 Quantity 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 target protein are highest when the culture-inducing temperature is 28 ℃,the methanol concentration is 1.0%,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.更多还原