巢湖学院学报2024，Vol.26Issue(6)：102-107,6.DOI:10.12152/j.issn.1672-2868.2024.06.013
拟南芥AtESD4基因的克隆及其体外原核表达体系的探索
Cloning of Arabidopsis AtESD4 Gene and Exploration of Its Prokaryotic Expression System in Vitro
摘要
Abstract
Current research on the biological functions of Arabidopsis AtESD4 primarily relies on esd4 mutants and in vivo experiments,while its recombinant protein expression and purification system remains unexplored.This study established a prokaryotic expression and purification system for A tESD4,identifying the optimal expression vector and induction time.The open reading frame(ORF)of A tESD4 was amplified from an Arabidopsis cDNA li-brary by PCR and cloned into prokaryotic expression vectors(pRSF-duet,pMAL-C2X,and pGEX-4T-1)using a one-step cloning method.Recombinant proteins were expressed in Escherichia coli,purified by affinity chromatog-raphy,and analyzed via SDS-PAGE to evaluate the effects of different vectors and induction times(8 h,16 h,and 24 h)on expression efficiency and purity.Results demonstrated that AtESD4 recombinant proteins predominantly formed inclusion bodies,with significantly higher abundance in bacterial precipitates than in supernatants.Among the three vectors,pMAL-C2X yielded AtESD4 fusion proteins with optimal quality and purity under 24 h induction,whereas pRSF-duet and pGEX-4T-1 showed inferior performance.关键词
拟南芥/AtESD4/基因克隆/体外蛋白重组/亲和层析Key words
Arabidopsis thaliana/AtESD4/gene cloning/in vitro protein recombination/affinity chromatography分类
生物科学引用本文复制引用
邢大伟,郑陶,吴浩男,马滢..拟南芥AtESD4基因的克隆及其体外原核表达体系的探索[J].巢湖学院学报,2024,26(6):102-107,6.基金项目
安徽省高等学校科学研究项目(项目编号:2024AH051350) （项目编号:2024AH051350）
安徽省高等学校省级质量工程项目(项目编号:2023sysx025) （项目编号:2023sysx025）
巢湖学院校级质量工程项目(项目编号:x24cjrhkc04) （项目编号:x24cjrhkc04）
