重组细胞色素P450 BM3 的表达及初步纯化研究.pdfVIP

I012 重组细胞色素P450 BM-3 的表达及初步纯化研究 黄俊，梅乐和，邓福华，林东强，姚善泾 （浙江大学化学工程与生物工程学系，杭州，310027 ） 摘 要 对重组细胞色素P450 BM-3 基因工程菌的培养条件进行了单因素优化，确定了较佳的培 养条件：接种量1%，装液量100mL/500mL，发酵培养基加入0.1 mg·L -1FeCl ，OD 达到 1.0 左 3 578 右时升温至42 ℃诱导，诱导时间为5 小时。用Resource Q 阴离子层析介质对菌体破胞液进行了 初步的纯化，通过ÄKTA explorer100 对其洗脱条件进行了优化，结果表明：0.3 mol·L -1 、0.5 mol·L -1 的两步NaCl 阶跃洗脱，P450 BM-3 纯度较高，活性收率为30.1%，纯化倍数为2.12 。 关键词 细胞色素P450 BM-3 高效表达 离子交换层析 STUDY ON THE FERMENTATION CONDITIONS AND PRIMARY PURIFICATION OF RECOMBINANT P450 BM-3 HUANG Jun, MEI Lehe, DENG Fuhua, LIN Dongqiang, YAO Shanjing (Department of Chemical and Biochemical Engineering, Zhejiang University,Hangzhou 310027, China) Abstract Cultivation conditions of the genetic engineering E. coli expressing P450 BM-3 were studied by single-factor optimization experiment. The results showed that higher expression of P450 BM-3 could be gained with inoculum quantity as 1% of the culture medium, inducing by a temperature shift from 37°C to 42°C quickly when its OD578 reached about 1.0 and keeping for 5 -1 hours. The addition of 0.1mg·L FeCl was also used to improve P450 BM-3 expression. Primary 3 purification of P450 BM-3 was investigated by one step Resource Q anion-exchange chromatography. By studying the elution condition of P450 BM-3 on ÄKTA explorer system., the result showed the purification factor and P450 BM-3 activity recovery were 2.12 and 30.1% through two-step 0.3 mol·L -1, 0.5 mol·L -1 NaCl elution. Keywords