重组人血清白蛋白-干扰素α2b融合蛋白在PichiaPink系统中的表达

Expression of Human Serum Albumin and Interferon α2b Fusion Protein in the PichiaPink System

目的:比较PichiaPink表达系统和巴斯德毕赤酵母GS115在表达外源蛋白方面的差异,对PichiaPink表达系统的潜在优点进行评价。方法:以重组人血清白蛋白-干扰素α2b融合蛋白(HSA-IFN-α2b)为报告蛋白,构建相关载体,分别转化PichiaPink系统菌株和巴斯德毕赤酵母GS115菌株,诱导HSA-IFN-α2b表达并测定表达水平;通过SDS-PAGE检测HSA-IFN-α2b在PichiaPink系统中的降解程度。结果:PichiaPink系统几乎所有的转化子都可以表达HSA-IFN-α2b,而GS115菌株只有60%的转化子能表达HSA-IFN-α2b;同一种Pink菌株中,整合有高拷贝数表达载体pPink-HC的菌株表达量高于整合有低拷贝数表达载体pPink-LC的菌株;Pink蛋白酶缺陷菌株在复杂培养基(YPD,BMMY)中HSA-IFN-α2b基本没有降解,而在合成培养基(BSM)中降解现象明显。结论:PichiaPink表达系统产生的转化子较GS115菌株产生的转化子易于筛选;PichiaPink系统蛋白酶缺陷菌株可明显减少外源蛋白降解。这些结果为利用PichiaPink表达系统高水平和大规模制备外源蛋白提供了实验依据。

Objective： The aim of this experiment was to compare the differences between the PichiaPink strain and Pichia pastoris GS115 strain,and further explore the potential advantages of the PichiaPink system.Methods： The recombinant expression vectors contained the fusion gene coding human serum albumin and interferon α2b fusion protein（HSA-IFN-α2b） were constructed,and were transformed them into the P.pastoris GS115 and the PichiaPink strain respectively.The fusion protein of HSA-IFN-α2b were expressed by methanol induction,and the expression level was evaluated.The SDS-PAGE analysis was used to detect the degradation of HSA-IFN-α2b in the P.pastoris GS115 and the PichiaPink strain.Results： Essentially all transformants in the PichiaPink system showed to express the objective protein of HSA-IFN-α2b,but only 60% transformants in the GS115 strain displayed to express the target protein.In the same kind strains of Pink,the expression levels of HSA-IFN-α2b were respectively compared,which discovered the strain that have integrated with the pPink-HC vector is higher expression than the strain that have integrated with the pPink-LC vector.The PichiaPink strains which had knockouted three proteases genes expressed the fusion protein of HSA-IFN-α2b to exhibit a little degradation in YPD and BMMY medium,but HSA-IFN-α2b degradation phenomenon showed very serious in BSM medium.Conclusion： The results have indicated to have an important value for high-level expression and large-scale production of secreted recombinant proteins in PichiaPink system.