摘要
甲酸脱氢酶(FDH,EC1.2.1.2)在工业生产中有重要的应用价值,工业上应用的FDH可以通过构建高水平表达重组FDH蛋白的基因工程菌生产,用分子生物学的方法检测重组蛋白的高效表达和积累操作繁杂,耗时耗力且需要破碎细胞。为了寻找一种简单快速,不需破碎细胞,且能实时检测FDH重组蛋白在基因工程菌中表达情况的方法,本研究应用单细胞激光拉曼光谱分析技术(LTRS),研究IPTG诱导不同时间后甲酸脱氢酶重组蛋白(FDH)在大肠杆菌细胞中的表达水平。结果表明,FDH的特征峰1004,1355,1455和1667cm-1随着IPTG诱导时间的延长而增强,说明在诱导培养过程中FDH重组蛋白在重组大肠杆菌细胞中表达并积累,这4个峰强的增加值所反映的FDH表达量与SDS-PAGE电泳分析结果一致。实验结果证明,LTRS是快速有效检测单个大肠杆菌活细胞体内重组FDH实时表达的一种非入侵方法。
The detection of the expression of formate dehydrogenase（FDH） recombination proteins in E.coli by molecular methods is time-consuming and hard-working and needs to destruct E.coli cells.To explore a simple and rapid method without cell destruction to detect the real-time expression of FDH recombination proteins in E.coli,Laser Tweezers Raman spectroscopy（LTRS） was used to investigate the recombinant protein expression of formate dehydrogenase（FDH） in the single living E.coli cell at different culture times following the induction with isopropyl thiogalactoside（IPTG）.The result showed that the characteristic peaks corresponding to the recombinant FDH protein were gradually enhanced with an in increase in IPTG induction time,indicating the expression and the accumulation of FDH protein in recombinant E.coli cells.This result is consistent with that obtained by the analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）.This evidence confirms that LTRS is an effectively method for detection of the real-time expression of FDH recombination proteins in the living single E.coli cell without cell destruction.
出处
《分析化学》
SCIE
EI
CAS
CSCD
北大核心
2012年第12期1845-1851,共7页
Chinese Journal of Analytical Chemistry
基金
国家自然科学基金(No.30970263)资助项目
关键词
大肠杆菌
甲酸脱氢酶
重组蛋白表达
单细胞
拉曼光谱
Escherichia coli
Formate dehydrogenase
Expression of recombinant protein
Single cell
Raman spectroscopy
