摘要
[目的]探究LpPEX5蛋白表达与纯化的最佳条件。[方法]从细叶百合的鳞茎中克隆出过氧化物酶体生物合成蛋白基因(LpPEX5),在详细分析该蛋白质同源氨基酸序列比对、进化树、保守区域,二级结构与三级结构预测等生物信息学后,构建到pQE-30原核表达载体上,转化大肠杆菌M15,通过SDS-PAGE凝胶电泳对不同诱导时间、诱导温度、IPTG浓度影响蛋白的表达量进行研究。利用亲核层析树脂纯化获得融合蛋白。[结果]成功克隆出LpPEX5基因,构建pQE-LpPEX5原核表达载体并且诱导和纯化出LpPEX5蛋白。[结论]LpPEX5蛋白的最佳诱导条件为30℃,1 mmol/L IPTG诱导5h。该研究为分析LpPEX5蛋白的活性、验证蛋白间的相互作用等后续试验研究奠定了基础。
[Objective]To explore the optimal conditions of expression and purification of Lp PEX5 protein.[Method]The LpPEX5 gene was cloned from the bulb of Lilium pumilum.After detailed analysis of the homologous amino acid sequence of LpPEX5,the phylogenetic tree,the conserved region,the secondary structure and the tertiary structure of Lp PEX5,and other bioinformatics,the recombinant plasmid p QE-30-Lp PEX5 was constructed and transformed into E.coli M15.SDS-PAGE electrophoresis was used to study the effects of induction time,induction temperature and IPTG concentration on the expression of the protein.Purification of fusion protein by nucleophilic chromatography resin.[Result]The Lp PEX5 gene was successfully cloned,the p QE-Lp PEX5 prokaryotic expression vector was constructed,and the Lp PEX5 protein was induced and purified.[Conclusion]Lp PEX5 protein was induced at 30℃for 5 hours by 1 mmol/L IPTG.This study laid a foundation for further study on the activity of Lp PEX5 protein and the interaction between Lp PEX5 protein.
作者
徐阳
何好
朱国庆
陈诗雅
金淑梅
XU Yang;HE Hao;ZHU Guo-qing;CHEN Shi-ya;JIN Shu-mei(Key Laboratory of Saline-alkali Vegetation Ecology Restoration,Ministry of Education,College of Life Sciences,Northeast Forestry University,Harbin 150040,China)
出处
《生物技术》
CAS
2020年第5期417-423,448,共8页
Biotechnology
基金
中央高校基本科研业务费专项资金资助(2572020DY18)
黑龙江省自然科学基金联合引导项目(LH2019C011)。
