红色荧光蛋白在毕赤酵母中的高效表达

报道了红色荧光蛋白(DsRFP)在毕赤酵母中的高水平表达．利用PCR技术从YEpFLAG-1-DsRFP扩增出DsRFP编码序列．克隆到毕赤酵母胞内表达载体pPIC3．5K构建重组表达载体pPIC3．5K-DsRFP．电击转化进毕赤酵母．G418-RDB平板双重筛选后获得重组转化子．经MM／MD平板培养与PCR鉴定．重组子全部为HIS^ -MUT^ 表型．重组菌株诱导表达48h后获得了高效表达．摇瓶中表达水平达到细胞内总蛋白的12％．表达量达到1．8g／L．经硫酸铵分级沉淀，DE-AE-5PW阴离子交换柱层析与S-HyperD阳离子交换柱层析后获得电泳纯蛋白．说明我们建立的毕赤酵母表达应用平台具有高效表达外源蛋白的能力。

Over-expression of Recombinant Red Fluorescent Protein(DsRFP) in the Methylotrophic Yeast Pichia pastoris

This study firstly reported the DsRFP over-expressed in the methylotrophic yeast P.pastoris.The coding sequence of red fluorescent protein (DsRFP) was amplified through ploymerase chain reaction(PCR) and cloned into yeast expression vector pPIC3.5K to construct plasmid of pPIC3.5K- DsRFP.The plasmids were linearized and then transformed into P.pastoris GS115 by electroporation.All of the recombinant strains were HIS~+MUT~+ identified by PCR and cultivated on MM/MD plate.The results of inducing expression showed that the rDsRFP was over-expressed after being induced 48 hours,the rDsRFP protein expressed in recombinant strain was about 12% of the total cell protein,1.8g/L of rDsRFP protein was achieved in the shake flask.The rDsRFP was purified through the steps of ammonium sulfate precipitation,DEAE-5PW aion-exchange chromatography and S-HyperD cation-exchange chromatography.