CYP3A4基因原核表达质粒构建与诱导表达

Construction of Recombinant Prokaryotic Expression Plasmid of CYP3A4 Gene and High Expression Induced by IPTG

以质粒pCWNF14为模板,采用PCR技术扩增出CYP3A4基因,并将其接入pET-22b(+)、pET-28b(+)和pET-32a(+)载体中,经PCR测序鉴定后,转化Rosetta(DE3)2pLysS细菌,并用IPTG诱导表达.采用考马斯亮蓝染色的方法检测重组蛋白表达,3个表达重组质粒中,只有pET-32a(+)-CYP3A4可以表达目的蛋白;在低浓度IPTG(50μmol/L)诱导6h,所表达的CYP3A4蛋白约占细菌总蛋白的40%,且该蛋白不溶于8mol/L的尿素,而溶于去污剂10mmol/L3-((3-Cholamidopropyl)dimethylammonio propanesulfonic acid(CHAPS))和0.3%lauroyl sarcosine(SKL).成功地使CYP3A4基因在原核系统中高效表达.

The CYP3A4 gene was amplified by method of PCR technique from the template of pCWNF14 plasmid, then was cloned into pET-22b（ ＋ ）, pET-28b（ ＋ ）, and pET-32a（ ＋ ） vector and identified with PCR and sequencing. The recombinant expression plasmid containing CYP3A4 gene was transformed into Rosetta （DE3） 2pLysS and target protein expression was induced by IPTG. Only recombinant plasmid of pET-32a（ ＋ ） -CYP3A4 could be induced by IPTG and express CYP3A4 protein. After 6 h induction in the concentration of 50μmol/L, the recombinant CYP3A4 protein accounted for 40% of total Rosetta（DE3） 2pLysS proteins detected by coomassie brilliant blue staining. The expressed target protein could be dissolved in 10 mmol/L 3- （3-Cholamidopropyl） dimethylammonio propanesulfonic acid （CHAPS） and 0.3 % lauroyl sarcosine （SKL）, but not in 8 mol/L urea. The recombinant CYP3A4 plasmid was successfully cloned and effectively expressed in Rosetta （ DE3 ） 2pLysS.