生物技术通报2016，Vol.32Issue(11)：130-136,7.DOI:10.13560/j.cnki.biotech.bull.1985.2016.11.015
极端嗜酸热古菌 Acidianus manzaensis 膜蛋白提取方法的建立及应用
Establishment of Extraction Methods of Membrane Proteins from Extremely Thermoacidophilic Acidianus manzaensis and Its Preliminary Application
摘要
Abstract
Using Acidianus manzaensis as research object,the methods of extracting the membrane proteins were explored and optimized,then the optimized methods were used to exact the membrane proteins of the bacterium cultured on elemental sulfur(S0)or ferrous iron(Fe2+)as energy substrates,and the differentiated expressions of the extracted membrane proteins from 2 substrates were analyzed by SDS-polyacrylamide gel electrophoresis(SDS-PAGE). The uncorrelated extracellular proteins were firstly removed by incubating the cells in 80℃ for 60-70 min. Then the extraction effects of different extraction reagents(Triton X-110,SDS and Triton X-114)on the membrane proteins were comparatively studied,and the results indicated that Triton X-114 was better than others and the optimal concentration of Triton X-114 was 10%(w/v). Similarly,the precipitation effects of different protein precipitation reagents(trichloroacetic acid(TCA),acetone, TCA/acetone,methyl alcohol and ethyl alcohol)on the proteins were comparatively studied,and the results indicated that the precipitation by TCA/acetone was the worst,as TCA/acetone caused the loss of low-molecular weight proteins ;while there were no significant differences by others. Compared comprehensively,the most commonly used acetone was chosen as the precipitation reagent in the extraction of membrane. Finally,the membrane proteins of A. manzaensis cultured on S0 and Fe2+ were extracted via the optimized traction method and comparatively analyzed by SDS-PAGE. The results showed that the protein bands with 35.6 kD and 16.8 kD only appeared in A. manzaensis cultured on S0, which was probably related to sulfur oxidation of this strain. The protein bands with 72 kD and 26 kD were significantly up-regulated in A. manzaensis cultured on Fe2+comparedwith on S0,indicating that the protein may be related to iron oxidation of this strain.关键词
Acidianus manzaensis/膜蛋白提取/SDS-PAGE/铁氧化/硫氧化Key words
Acidianus manzaensis/membrane proteins extraction/SDS-PAGE/iron oxidation/surfur oxidation引用本文复制引用
杨云,刘红昌,夏金兰,马亚龙,聂珍媛..极端嗜酸热古菌 Acidianus manzaensis 膜蛋白提取方法的建立及应用[J].生物技术通报,2016,32(11):130-136,7.基金项目
国家自然科学基金项目 （）
