【作者】 孔宁； 周余来； 王毅； 宿晓云； 王芳； 丛中一； 苏曼曼； 侯宜； 颜炜群；

【Author】 KONG Ning,ZHOU Yu-lai,WANG Yi,SU Xiao-yun,WANG Fang,CONG Zhong-yi,SU Man-man, HOUYi,YANWei-qun~* (Department of Regenerative Medicine,School of Pharmaceutical Sciences of Jilin University, Changchun,130021,China )

【机构】 吉林大学药学院再生医学系；

【摘要】 目的研究重组人抑瘤素M（rhOSM）在毕赤酵母中分泌表达的条件及rhOSM的纯化方法。方法利用已筛选出来的rhOSM毕赤酵母高表达菌株,在实验室条件下,利用5 L摇瓶发酵表达rhOSM。对发酵液上清分别应用SP Sepharose XL阳离子交换层析和Source^TM30 RPC反向疏水层析进行纯化,对纯化产物进行SDS-PAGE分析及Western Blotting鉴定。结果在5 L摇瓶的规模,pH 6.0的条件下,以0.5%甲醇诱导表达72 h,rhOSM表达量达到高峰。经SP Sepharose XL阳离子交换层析和Source^TM30 RPC反向疏水层析纯化,rhOSM回收率为55%,纯度可达94%。结论确定了在5 L摇瓶规模,rhOSM分泌表达的最佳条件。更多还原

【Abstract】 OBJECTIVE To explore the method of secretory expression and purification of recombinant human oncostatin M（rhOSM） in Pichia pastoris.METHODS Under laboratory conditions,rhOSM was expressed in 5 L shake flask with Pichia pastoris engineering strain of highly effective expression of rhOSM. rhOSM was purified by SP Sepharose XL cation exchange chromatography and Source^TM 30 RPC reverse hydrophobic interaction chromatography.The supernatant were analyzed by SDS-PAGE and Western blotting. RESULTS After 0.5%methanol induction for about 72 h at pH6.0,the expression level of rhOSM peaked.The cell-free supernatants collected were purified by SP Sepharose XL cation exchange chromatography and Source^TM 30 RPC reverse hydrophobic interaction chromatography,which resulted in a recovery of 55%and a purity of 94%.CONCLUSION In the 5 L flask scale,the growth conditions of the transformant strain of rhOSM were optimized.更多还原