摘要
为拟南芥细胞色素P450 94B3-N25的优化表达和纯化,利用原核表达技术,成功在大肠埃希菌内表达了拟南芥细胞色素P450 94B3-N25。首先,用热激转化方法将载体pMAL-c2x-IPN-CYP94B3-N25转入Rosseta,利用菌落PCR挑选阳性克隆,设置交叉试验,摸索最适表达温度和时间表达目的蛋白,并在450nm处利用分光光度法测定菌液里有目的蛋白的表达。破碎细胞后,提取细胞总蛋白,SDS-PAGE和Western blot鉴定结果显示目的蛋白表达成功,但蛋白质变性。
Arabidopsis thaliana cytochrome P450 94 B3-N25 was successfully expressed in Escherichia coli using prokaryotic expression technology.pMAL c2 x-IPN-94 B3-N25 was transformed into Rosseta by the method of heat shock,and the positive clones were screened by PCR,with the help of crossing experiment and spectrophotometry,the best temperature and time to express purpose protein were optimized,and the protein at 450 nm in bacteria liquid was detected.We gained the total protein with cell disruption,the results of SDS-PAGE and Western blot showed the protein expressed successfully,but denatured.
引文
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