重组铜绿假单胞菌外毒素A工程菌发酵及表达产物的纯化

目的建立稳定、高效表达重组铜绿假单胞菌外毒素A(rEPA)的工程菌发酵及表达产物纯化工艺。方法规模化发酵表达rEPA的重组大肠杆菌rPE553D,离心收集菌体,渗透压调解使菌体周质间隙蛋白释放后,高速离心收集蛋白溶液。经DEAESepharoseFF、PhenylSepharose6FF疏水层析和SOURCE30Q强离子交换层析,超滤浓缩纯化rEPA。用HPLC、SDS-PAGE和Westernblot等方法检定生化和免疫学特性,并用小鼠和Vero细胞检定毒性。结果每55L培养基中rEPA产量超过4g,纯度在95%以上,细胞毒性降低了至少32000倍。其余各项指标均符合《中国生物制品规程》要求。结论已建立了收率高、纯度好、稳定、适合规模化生产rEPA的工艺。

Fermetation of Engineering E. coli Strain Expressing Recombinant Pseudomonas aeruginosa Exotoxin A and Purification of Expressed Product

Objective To develop a procedure for fermentation of engineering E.coli strain stably and highly expressing recombinant Pseudomonas aeruginosa exotoxin A(rEPA) and for purification of expressed product.Methods Culture the engineering E.coli rPE553D for expression of rEPA in a large scale by fermentation,harvest the bacteria by centrifugation,release the protein by adjusting osmotic pressure,then collect the supernatant by high speed centrifugation and purify by DEAE Sepharose FF,Phenyl Sepharose 6FF and SOURCE 30Q chromatography and ultrafiltration.The purified rEPA was identified by HPLC,SDS-PAGE and Western blot and tested for cytotoxicity by mouse and Vero cell methods.Results The yield of rEPA in every 55 L of medium was more than 4 g,and its purity was more than 95%,however,its cytotoxicity decreased by 32 000 folds.All the other indexes of rEPA met Chinese Requirements for Biologics.Conclusion A stable procedure for large-scale production of rEPA was developed.Both the yield and purity of prepared rEPA were high.