人睫状神经营养因子在大肠杆菌和毕赤酵母中的表达和生物活性测定  

Biological Activity Studies of Human Ciliary Neurotrophic Factor expressed in E.coli and Pichia pastoris

作　　者：张惠堂[1] 颜志强[1] 蔡兴锋[1] 杨胜利[1] 龚毅[1] 

机构地区：[1]中国科学院上海生命科学院上海生物工程研究中心,上海200233

出　　处：《中国生物化学与分子生物学报》2005年第3期427-430,共4页Chinese Journal of Biochemistry and Molecular Biology

摘　　要：The recombinant human ciliary neurotrophi factor(hCNTF)expressed in E.coli aggregatedas inclusion bodies and refolding procedure was necessary to obtine the active protein.To overcome the disadvantage,we cloned hcntf gene into yeast expression plasmid pPIC9K and collected the plasmid pPIC9K-hcntf.Plasmid pPIC9K-hcntf was transformed into yeast Pichia pastoris GS115,and screened on G418-SD plates.The transformants with high copies of hcntf gene were inoculated into BMMY media and induced with 0.5% methanol.The recombinant hCNTF was secreted into the media.The amount of hCNTF in the supernatant was about 10 mg/L when incubated in the conical flasks and reached up to 60 mg/L under fed-batch condition in 15 L fermentator.The recombinant hCNTF expressed in E.coli was renatured as the control.The neonatal rat dorsal root ganglion assay showed that proteins expressed in both systems have the activity of promoting the growth of neuron axons.The phenomenon can be observed with only 3 μg hCNTF expressed in yeast present,which indicates that hCNTF was successfully expressed in Pichia pastoris and has a relatively high activity.The recombinant human ciliary neurotrophi factor(hCNTF)expressed in E.coli aggregatedas inclusion bodies and refolding procedure was necessary to obtine the active protein.To overcome the disadvantage,we cloned hcntf gene into yeast expression plasmid pPIC9K and collected the plasmid pPIC9K-hcntf.Plasmid pPIC9K-hcntf was transformed into yeast Pichia pastoris GS115,and screened on G418-SD plates.The transformants with high copies of hcntf gene were inoculated into BMMY media and induced with 0.5% methanol.The recombinant hCNTF was secreted into the media.The amount of hCNTF in the supernatant was about 10 mg/L when incubated in the conical flasks and reached up to 60 mg/L under fed-batch condition in 15 L fermentator.The recombinant hCNTF expressed in E.coli was renatured as the control.The neonatal rat dorsal root ganglion assay showed that proteins expressed in both systems have the activity of promoting the growth of neuron axons.The phenomenon can be observed with only 3 μg hCNTF expressed in yeast present,which indicates that hCNTF was successfully expressed in Pichia pastoris and has a relatively high activity.

关 键 词：人睫状神经营养因子 大肠杆菌 毕赤酵母 基因表达 生物活性 

分 类 号：R338.1[医药卫生—人体生理学] Q786[医药卫生—基础医学]