摘要
以米黑根毛霉(Rhizomucor miehei)为出发菌株,采用UNIQ-10柱式Trizol总RNA抽提试剂盒提取其总RNA,反转录-聚合酶链式反应(RT-PCR)扩增出米黑根毛霉脂肪酶(RML)结构基因。构建真核重组表达质粒RML-pPIC9K,电转化His+缺陷型巴斯德毕赤酵母(Pichia pastoris)GS115,利用MD-MM平板及PCR方法筛选和鉴定出重组子,进行甲醇诱导表达。结果表明,RML基因能够在巴斯德毕赤酵母中实现高效表达。重组子发酵液经SDS-PAGE分析显示获得了与预期大小(约39 kD)一致的蛋白表达特异条带,用NaOH滴定法测其活性,酶活可达84 U/mL。
Lipase gene cDNA fragment from Rhizomucor miehei（RML）was amplified by RT-PCR method.Expression vector RMLpPIC9K containing lipase gene was constructed and the gene was expressed in His4 mutant Pichia pastoris yeast GS115.Recombinant Pichia strains were obtained by minimal olive oil-methanol plates screening and confirmed by PCR.The expression product of RML gene was analysis by SDS-PAGE,and molecular weight of the expressed protein was estimated to be 39 kD.The activity of lipase was up to 84 U/mL determined by the method of titration of NaOH.The result indicated that RML gene was functionally overexpressed inPichia pastoris.
出处
《生物技术通报》
CAS
CSCD
北大核心
2013年第6期122-127,共6页
Biotechnology Bulletin
基金
“十二五”农村领域国家科技计划课题(2011BAD22B05)
中科院广州能源所博士启动基金项目(y107rb1001)
