拟南芥FtsH12蛋白原核表达载体构建及表达条件优化.doc

拟南芥FtsH12原核表达载体构建及表达条件优化 * 1中国科学院青岛生物能源与过程研究所，青岛，266101；2中国科学院大学，北京，100049 3 中国科学院天津工业生物技术研究所，天津，300308 *通讯作者，zhang_dy@tib.cas.cn 摘要FtsH蛋白酶在植物叶绿体生物发生及胚胎发育过程中起到重要的作用。前期实验我们发现在拟南芥中AtFtsH12基因敲除导致胚胎致死，为研究AtFtsH12的生物学功能，本文成功构建了包含拟南芥FtsH12蛋白两个主要功能域的原核表达载体pEASY-E1-AtFtsH12s并通过正交快速优化诱导表达条件增目的蛋白的表达量。选取诱导时机、诱导剂浓度、诱导温度和诱导时间4个外部因素采用四因素三水平正交设计AtFtsH12s的表达条件。统计分析结果诱导时机、诱导温度和诱导时间，IPTG浓度对蛋白表达的影响不显著最佳条件为诱导菌液初始浓度OD600值0.4，诱导IPTG浓度0.6 m mol/L，诱导温度30 ℃，诱导时间5 h为后期的纯化蛋白以及酶活性实验奠定了基础 关键词AtFtsH12蛋白原核表达正交条件优化 Construction of prokaryotic expression vector pEASY-E1-AtFtsH12s and optimization of AtFtsH12 protease expression ZWen Zenwen3 Ma Yubin1 ZhuMing1 HeGuo1 Zhang Donyuan1,3 1 Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266101; 2 University of Chinese Academy of sciences, Beijing; 3 Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 * Corresponding author, Zhang Donyuan, zhang_dy@tib.cas.cn Abstract FtsH proteases play important role in chloroplast biogenesis and embryonic development. In previous study, we found that AtFtsH12 knockout result in embryo lethality, to 基金项目:本研究由国家自然基金面上项目 investigate its function, a pEASY-E1-AtFtsH12s prokaryotic expression vector containing ATPase and Zn2+-binding domain of AtFtsH12 protease was constructed in this study. Subsequently, its recombinant protein expression was identified by SDSand Western blot. Further, the expression of AtFtsH12s protease was optimized by orthogonal analysis with four influence factors on three levels including cell density, inductor concentration, induction temperature, and induction time. The expression level of AtFtsH12s was used as the evaluation parameter in different conditions. The result showed that the main influence factors were cell density, induction temperature and induction time. The optimum condition of maximum expression was OD600 0.4, 0.6 m mol/L IPTG, 30 ℃ and 5 h. This study laid the foundation for