摘要
为获得ＩＧＦ－Ⅱ的高效表达，构建了其多拷贝、分泌型表达载体：含有５个串联的ＩＧＦ－Ⅱ表达单元；由受甲醇诱导的醇氧化酶启动子控制表达；利用啤酒酵母α因子的前导肽引导分泌．线形化表达载体转化Ｐ．ｐａｓｔｏｒｉｓ蛋白酶缺陷型菌株后，筛选到有ＩＧＦ－Ⅱ表达和分泌的阳性转化子；进一步优化表达、培养条件后，ＩＧＦ－Ⅱ在高密度发酵上清中的产量可达６０ ｍ ｇ／Ｌ．对Ｐ．ｐａｓｔｏｒｉｓ产生的ｒｈＩＧＦ－Ⅱ的性质分析表明，其具有正确的分子量，Ｎ 端和较好的生物活性．
To increase the expression level of IGF Ⅱ,an expression vector containing a tandem of 5 IGF Ⅱ expression cassettes with alcohol oxidase(AOX1)promoter was constructed.The gene encoding the mature form of IGF Ⅱ was fused in frame to the S.cerevisiae α mating factor leader sequence,that is,the secretion of IGF Ⅱ depended on the secretion signal sequence of α factor.After transformation of P.pastoris proteinase deficient strain with linearized expression vector,the transformants expressed and secreted IGF Ⅱ to the growth medium were screened.Then the culture of expression strain was optimized and scaled up,the yield in the supernatant of high biomass fermentation culture was at the level of 60 mg/L.Further analyses of rhIGF Ⅱ produced in P.pastoris show that it has correct M r,N terminal amino acid sequence and high bioactivity.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
1999年第6期893-898,共6页
Chinese Journal of Biochemistry and Molecular Biology
关键词
甲醇营养型酵母
分泌
IGF-Ⅱ
表达
性质
Insulin like growth factor Ⅱ(IGF Ⅱ),P.pastoris,High level expression,Secretion
