摘要
从抗生链霉菌(Streptomyces antibioticus)中提取出目的基因phs,构建基于质粒pET28a的诱导型表达载体pET28a-phs。以E.coli为表达宿主,将重组质粒转化入宿主菌之后获得能够高效表达吩噁嗪酮合成酶(PHS)的重组菌,经SDS-PAGE验证在72.1 kD处有明显蛋白条带。通过调整可能影响表达的参数获得PHS在大肠杆菌中的最优表达条件。最终优化后的表达条件是最适Cu2+浓度为1.5 mmol/L,最适细菌培养温度为37℃,最适pH值为7.0,在OD600为0.6,时加入终浓度为1.0 mmol/L的IPTG为最佳诱导条件,30℃诱导16 h为最佳诱导时间。
It was to optimize and increase the expression of per unit of laccase in E.coli expression system BL21.Laccase-dependent Phenoxazinone synthase（phs）from Streptomyces antibioticus was cloned into Escherichia coli and expressed,through constructing the recombinant plasmid pET-phs,which was then translated into the host E.coli BL21.Furthermore.expression of PHS protein with 72.1 kD was detected by SDS-PAGE.Factors which might effect the expression were optimized on the basis of process.The optimal parameter of expressing laccase is：culture the E.coli with the recombinant in 37℃ pH 7.0 LB culture medium with Cu2＋ 1.5 mmol/L by using horizontal swing bed 180 r/min.While OD600 goes to 0.6 adding IPTG to final concentration 1.0 mmol/L,collects the bacterial precipitation by centrifugation after the E.coli has been induced for 16 hours in 30℃.
出处
《生物技术通报》
CAS
CSCD
北大核心
2011年第4期176-180,共5页
Biotechnology Bulletin
基金
国家自然科学基金资助项目(20906048)
