大肠杆菌外膜蛋白的分离及其双向电泳图谱的建立  

Rapid and Efficient Extraction of Outer Membrane Proteins from Escherichia coli and Establishment of the Two-Dimensional Electrophoresis Maps

作　　者：夏金兰[1] 欧阳叙东[1] 张成桂[1] 彭安安[1] 王晶[1] 

机构地区：[1]中南大学资源加工与生物工程学院生物冶金教育部重点实验室,湖南长沙410083

出　　处：《现代生物医学进展》2009年第2期201-204,215,共5页Progress in Modern Biomedicine

基　　金：国家973计划"微生物冶金的基础研究"项目(2004CB619201);国家自然基金创新研究群体科学基金项目(50621063);国家自然科学基金项目(50674101)

摘　　要：本文利用温度诱导的两相分离萃取技术选择性分离未经机械破碎的大肠杆菌细胞外膜蛋白,研究Triton X-114的浓度及处理时间对提取外膜蛋白的影响。实验结果表明,Triton X-114的使用浓度和作用时间均显著影响外膜蛋白的提取效率。SDS-PAGE结果表明不同Triton X-114的使用浓度和作用时间只是影响了外膜蛋白的提取效率而对外膜蛋白提取的种类没有影响。实验发现8%的Triton X-114处理3小时为最佳分离条件,分离得到的样品可用于双向电泳分析。通过对比实验发现样品裂解液中包含低浓度的Tris是外膜蛋白双向电泳成功的关键因素,CHAPS与ASB-14或NP-40结合使用可显著提高外膜蛋白的溶解能力,缩短聚焦时间,从而优化了大肠杆菌外膜蛋白双向电泳技术体系,建立了其双向电泳图谱。The outer membrane proteins （OMPs） are important part in the Gram-negative bacteria,which are responsible for mass transport, signal recognition and cell attachment. Extraction of outer membrane proteins is the initial step for their further investigation. The traditional extraction methods, however, are time-consuming,complex and difficult to differentiate the outer membrane proteins from the cytoplasm membrane proteins. In this paper, we reported a rapid and efficient method based on temperature-induced phase separation technique to extract selectively the outer membrane proteins fi-om the unbroken Escherichia coli cells.By systematic experimental essaying on the effects of Triton X-114 concentrations and the treatment time on the extraction of OMPs,we found that 8 % of Triton X-114 treating for 3 hours was the optimal extraction condition. The extracted samples were separated by two-dimensional electrophoresis （2-DE）.The 2-DE results indicated that lysis buffer containing low concentration Tris played a key role for the success of 2-D,and that CHAPS combined with ASB-14 orNP-40 could increase the outer membrane proteins solubilization and decrease the isoelectric focusing time.Thus, an efficient 2-D separation system for Escherichia coli outer membrane proteins was established.

关 键 词：大肠杆菌 外膜蛋白 TritonX-114 双向电泳 

分 类 号：Q591.2[生物学—生物化学]