极端嗜酸热古菌Acidianus manzaensis膜蛋白提取方法的建立及应用

doi:10.13560/j.cnki.biotech.bull.1985.2016.11.015

生物技术通报 ›› 2016, Vol. 32 ›› Issue (11): 130-136.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

杨云¹, 刘红昌¹, 夏金兰^{1, 2}, 马亚龙¹, 聂珍媛^{1, 2}

1. 中南大学资源加工与生物工程学院,长沙 410083;
2. 中南大学教育部生物冶金重点实验室,长沙 410083

收稿日期:2016-03-07 出版日期:2016-11-25 发布日期:2016-11-11
作者简介:杨云,男,研究方向：生物冶金;E-mail：yysgamesemail@163.com
基金资助:
国家自然科学基金项目（51274257,U1232103）

Establishment of Extraction Methods of Membrane Proteins from Extremely Thermoacidophilic Acidianus manzaensis and Its Preliminary Application

YANG Yun¹, LIU Hong-chang¹, XIA Jin-lan^{1, 2}, MA Ya-long¹, NIE Zhen-yuan^{1, 2}

1. School of Minerals Processing and Bioengineering,Central South University,Changsha 410083;
2. Key Laboratory of Biometallurgy of Ministry of Education,Central South University,Changsha 410083

Received:2016-03-07 Published:2016-11-25 Online:2016-11-11

摘要/Abstract

摘要： 以万座嗜酸两面菌（Acidianus manzaensis）为研究对象,探索并优化其膜蛋白提取方法,以优化后的方法提取该菌分别以单质硫（S⁰）和亚铁（Fe²⁺）为能源底物进行生长时的膜蛋白质,并进行聚丙烯酰胺凝胶电泳（SDS-PAGE）研究膜蛋白在两种能源底物培养下的表达差异。首先通过80℃水浴60-70 min对A. manzaensis胞外黏附蛋白进行初步分离。其次比较了不同提取剂（Triton X-110,SDS和Triton X-114）对膜蛋白的提取效果,结果表明Triton X-114的提取效果较好,其最佳浓度为10%（w/v）;比较了不同沉淀剂（三氯乙酸（TCA）,丙酮,三氯乙酸/丙酮,甲醇和乙醇）对膜蛋白质沉淀的效果,结果表明TCA/丙酮的沉淀效果最不理想,会导致沉淀后低分子量蛋白发生缺失,而其他几种没有明显差别,综合比较选择较常用的丙酮作为膜蛋白提取的沉淀剂。最后基于优化后膜蛋白提取方法,分别对S⁰和Fe²⁺培养的A. manzaensis膜蛋白质进行提取及SDS-PAGE,结果发现分子量为35.6 kD 和16.9 kD的蛋白只在A. manzaensis以S⁰生长时出现,表明这些蛋白质很可能在A. manzaensis硫氧化中发挥了重要作用;分子量为72 kD 和26 kD的蛋白质在A. manzaensis以Fe²⁺生长时比其以S⁰生长时显著表达上调,表明此蛋白可能与A. manzaensis铁氧化相关。

关键词: Acidianus manzaensis, 膜蛋白提取, SDS-PAGE, 铁氧化, 硫氧化

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.

Key words: Acidianus manzaensis, membrane proteins extraction, SDS-PAGE, iron oxidation, surfur oxidation

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.

YANG Yun, LIU Hong-chang, XIA Jin-lan, MA Ya-long, NIE Zhen-yuan. Establishment of Extraction Methods of Membrane Proteins from Extremely Thermoacidophilic Acidianus manzaensis and Its Preliminary Application[J]. Biotechnology Bulletin, 2016, 32(11): 130-136.

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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

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