活性污泥总蛋白提取方法的比较
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摘要
有效提取总蛋白是活性污泥蛋白质组学研究的第一步,由于活性污泥中含有腐殖质以及其他复杂的有机物质,因此常规的提取方法均难以获得高质量的蛋白质。文章考察了SDS-酚-TCA法、SDS-酚法、柠檬酸钠-酚法、SDS提取法和SDS震荡提取法抽提活性污泥总蛋白的效果,比较了蛋白得率,并通过SDS-PAGE对蛋白的质量进行了检测。结果表明,5种方法均可从活性污泥中提取蛋白质,但5种方法在提取的蛋白的得率和质量上存在差异,其中采用SDS-酚法抽提的蛋白质效果最好,以该方法提取的蛋白质得率最高并且蛋白条带最多。该文为活性污泥总蛋白的提取提供了一种有效的方法。
Isolation of high-quality protein from activated sludge is the first step to study activated sludge metaproteomics.Activated sludge is rich in humic substances and other organic substances which may cause low yield of protein during protein extraction with the conventional method. Five protein extraction methods such as SDS-phenol-TCA extract,SDS-phenol extract,citrate-phenol extract,SDS extract and SDS-shaking-extract were compared and investigated. Yield of the extracted protein and protein quality were tested by SDS-PAGE. The results showed that all these methods can extract certain protein from activated sludge but with different yield and quality,SDS-phenol extract obtained the highest total protein yield and bands. This study provides an efficient method for extracting total protein from activated sludge.
Isolation of high-quality protein from activated sludge is the first step to study activated sludge metaproteomics.Activated sludge is rich in humic substances and other organic substances which may cause low yield of protein during protein extraction with the conventional method. Five protein extraction methods such as SDS-phenol-TCA extract,SDS-phenol extract,citrate-phenol extract,SDS extract and SDS-shaking-extract were compared and investigated. Yield of the extracted protein and protein quality were tested by SDS-PAGE. The results showed that all these methods can extract certain protein from activated sludge but with different yield and quality,SDS-phenol extract obtained the highest total protein yield and bands. This study provides an efficient method for extracting total protein from activated sludge.
引文
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[2]Amman R I,Ludwig W,Schleifer K H.Phylogenetic identification and in situ detection of individual microbial cells without cultivation[J].Microbiol Rev,1995,59:143-169.
[3]孟飞,俞春娜,王秋岩,等.宏基因组与宏基因组学[J].中国生物化学与分子生物学报,2010,2(2):116-120.Meng Fei,Yu Chunna,Wang Qiuyan,et al.Metagenome and metagenomics[J].Chinese Journal of Biochemistry and Molecular Biology,2010,2(2):116-120.(in Chinese)
[4]刘文干,吴云,崔中利,等.元蛋白质组学在土壤微生物生态功能研究中的应用[J].土壤学报,2012,49(6):1218-1226.Liu Wengan,Wu Yun,Cui Zhongli,et al.Application of metaproteomics in studying ecological function of soil microorganisms[J].Acta Pedologica Sinica,2012,49(6):1218-1226.(in Chinese)
[5]Kuhn R,Benndorf D,Rapp E,et al.Metaproteome analysis of sewage sludge from membrane bioreactors[J].Proteomics,2011,11(13):2738-2744.
[6]Abram F,Gunnigle E,O'Flaherty V.Optimisation of protein extraction and 2-DE for metaproteomics of microbial communities from anaerobic wastewater treatment biofilms[J].Electrophoresis,?2009,30(23):4149-4151.
[7]Park C,Helm RF.Application of metaproteomic analysis for studying extracellular polymeric substances(EPS)in activated sludge flocs and their fate in sludge digestion[J].Water Sci Technol,2008,57(12):2009-2015.
[8]Wilmes P,Wexler M,Bond P L.Metaproteomics provides functional insight into activated sludge wastewater treatment[J].PLo S One,2008,3(3):1778.
[9]Damerval C,Vienne D,Zivy M,et al.Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins[J].Electrophoresis,1986,7(1):52-54.
[10]Carpentier S C,Witters E,Laukens K,et al.Preparation of protein extracts from recalcitrant plant tissues:an evaluation of different methods for two-dimensional gel electrophoresis analysis[J].Proteomics,2005,5(10):2497-2507.
[11]康俊梅.低温胁迫下野牛草生理生化响应及蛋白质组学研究[D].北京:中国农业科学院,2008:44-50.Kang Junmei.Study on the Physiological and Biochemical Response and Differentially Expressed Proteome under Cold Stress in Buffalograss[D].Beijing:Chinese Academy of Agricultural Science,2008:44-50.(in Chinese)
[12]Gu R S,Liu Q L,Chen X M,et al.An economical and effective method for two-dimensional electrophoresis of proteins of woody plant[J].Journal of Beijing Forestry University,1999,21(5):1-7.
[13]Herman E M,Helm R M,Jung R,et al.Genetic modification removes an immunodominant allergen from soybean[J].Plant Physiology,2003(132):36-43.
[14]Florence Abram,Eoin Gunnigle,Vincent O’Flaherty.Optimisation of protein extraction and 2-DE for metaproteomics of microbial communities from anaerobic wastewater treatment biofilms[J].Electrophoresis,2009(30):4149-4151.
[15]Rathsam C,Eaton RE,Simpson CL,et al.Two-dimensional fluorescence difference gel electrophoretic analysis of Streptococcus mutans biofilms[J].J Proteome Res,2005,4(6):2161-2173.
[16]Wang W,Vignani R,Scali M,et al.A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis[J].Electrophoresis,2006(27):2782-2786.
[17]Katharina M,Keiblingera M,Keiblinger,et al.Soil metaproteomics-comparative evaluation of protein extraction protocols[J].Soil Biology&Biochemistry,2012(54):14-24.
[18]Chen S,Rillig M,Wang W.Improving soil protein extraction for metaproteome analysis and glomalin-related soil protein detection[J].Proteomics,2009(9):4970-4973.
[19]Haibin Wang,Zhixing Zhang,Hui Li,et al.Characterization of metaproteomics in crop rhizospheric soil[J].Journal of Proteome Research,2011(10):932-940.
[20]Bradford M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding[J].Analytical Biochenmistry,1976(72):248-254.
[21]温洪宇,王璐,杨柳,等.乳品废水活性污泥总DNA提取方法的比较[J].环境科学与管理,2012,37(4):144-148.Wen Hongyu,Wang Lu,Yang Liu,et al.Comparison of different DNA extraction methods from dairy wastewater activated sludge[J].Environmental Science and Management,2012,37(4):144-148.(in Chinese)
[22]Giagnoni L,Magherini F,Landi L,et al.Soil solid phases effects on the proteomic analysis of Cupriavidus metallidurans CH34[J].Biology and Fertility of Soils,2012,48(4):425-433.
[23]Robe P,Nalin R,Carmela C,et a1.Extraction of DNA from soil[J].J Soil Biology,2003(39):183-190.