Acidiphilium DX1-1细胞膜蛋白质表达谱构建
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摘要
随着铁呼吸机制的深入研究,Acidiphilium sp.菌株的重要性也越来越被人们所重视。在生物冶金方面,利用Acidiphilium sp.菌株与其他微生物协同浸矿,可通过降低有机物对自养菌的毒害作用以及提供低pH环境而有效提高浸矿效率,并可再生Fe2+；在生物燃料方面,Acidiphilium sp可在低pH环境中做为生物燃料电池的正极生物催化剂。
本文通过提取Acidiphilium cryptum DX1-1菌株膜相关蛋白,利用SDS-PAGE将膜蛋白分开,之后利用胶内酶解技术,采用液相色谱—串联质谱技术先对酶解后的蛋白进行分离,分离后的肽片段直接进入质谱仪离子源进行一级和二级质谱的分析,构建Acidiphilium cryptum DX1-1菌株细胞膜相关蛋白质的表达图谱。通过Mascot数据库进行搜库比对,鉴定得到262个匹配蛋白。
对这262个蛋白输出信号及亚细胞定位进行预测,并对其功能进行分析和预测。这些已鉴定的这些已鉴定的蛋白中13.7%有Sec-tYPe信号肽,27.86%具有双精氨酸转运信号肽(Tat),18.32%的蛋白具有脂蛋白信号肽(Lipo),14.9%含有C末端GPI锚定信号序列的有,2.67%同时含有C端和N端锚定信号序列GPI锚定蛋白。统计等电点及分子量得知,蛋白质的pH主要分布在4～12之间,40%蛋白质的pⅠ小于7；60%蛋白质pⅠ大于7；27.48%蛋白pⅠ大于10。
与Acidiphilium cryptum DX1-1细胞壁蛋白比对,得到30个重复蛋白,包括3个能量代谢相关蛋白,8个运输与结合蛋白,2个蛋白质合成相关蛋白,2个蛋白质修饰相关蛋白,3个假定蛋白,8个细胞包被蛋白以及4个功能未知蛋白。
Acidiphilium cryptum DX1-1.细胞膜相关蛋白的表达图谱的建立,为以后各种环境中细胞膜相关蛋白的研究提供了有价值的基础性研究。从蛋白质组学角度探讨浸矿微生物铁呼吸相关的蛋白质,为微生物铁呼吸机理的研究提供一些依据。
With the reseach of iron respiration, Acidiphilium sp.has been paid more attention to,because of its iron respiration,. In the application of bioleaching
Acidiphilium sp. plays an important role during form ation of precipitated ores, which can cooperate with other microorganisms in bioleaching process.Via reducing the poisonous impact of organics to bioleaching orgnisms providing low pH enviroment, Acidiphilium sp.can effectively increase the efficiency of bioleaching.Nowadays,a microbial fuel cell has been developed using the acidophile, Acidiphilium cryptum, as the anode biocatalyst。
In this study, the cell membrane related proteins of Acidiphilium DX1-1. were extracted and separated by SDS-PAGE. And then,subjected in-gel digestion.The peptides,which were seperated by liquid chromatography,were directly subjected for online analysis by the coupled tandem mass spectrometry. With Mascot search engine,262 identification proteins were identified.
The protein export signal, subcellular location and function of each identified protein were predicted. Among these identified proteins, 13.7% possess export of the Sec type,27.86% show a twin arginine translocation (Tat) signal,18.32% have lipoprotein signal peptides.14.9% have a C-terminal GPI-anchor signal sequence,and 2.67% have both C-and N-terminal signal sequences. According to satistics assay, the molecular weights of these proteins are distributed in the range of 4KDa to 120KDa;40% of which show the pI values below 7,60% higher than 7, and 27.48% over 10.
30 identified proteins in this study are coshared between cell wall and cellmembrane enrichments, which include 3 energy metabolism related proteins,8 transport and binding proteins,2 Protein synthezing related proteins,2 modification related proteins,8 Cell envelope related proteins, and 4 proteins with unknown function.
The Acidiphilium DX1-1 membrane proteomics in this study will provide solide foundation in clarifying the mechanisms of iron respiration of Acidiphilium DX1-1 under different enviroments.
本文通过提取Acidiphilium cryptum DX1-1菌株膜相关蛋白,利用SDS-PAGE将膜蛋白分开,之后利用胶内酶解技术,采用液相色谱—串联质谱技术先对酶解后的蛋白进行分离,分离后的肽片段直接进入质谱仪离子源进行一级和二级质谱的分析,构建Acidiphilium cryptum DX1-1菌株细胞膜相关蛋白质的表达图谱。通过Mascot数据库进行搜库比对,鉴定得到262个匹配蛋白。
对这262个蛋白输出信号及亚细胞定位进行预测,并对其功能进行分析和预测。这些已鉴定的这些已鉴定的蛋白中13.7%有Sec-tYPe信号肽,27.86%具有双精氨酸转运信号肽(Tat),18.32%的蛋白具有脂蛋白信号肽(Lipo),14.9%含有C末端GPI锚定信号序列的有,2.67%同时含有C端和N端锚定信号序列GPI锚定蛋白。统计等电点及分子量得知,蛋白质的pH主要分布在4～12之间,40%蛋白质的pⅠ小于7；60%蛋白质pⅠ大于7；27.48%蛋白pⅠ大于10。
与Acidiphilium cryptum DX1-1细胞壁蛋白比对,得到30个重复蛋白,包括3个能量代谢相关蛋白,8个运输与结合蛋白,2个蛋白质合成相关蛋白,2个蛋白质修饰相关蛋白,3个假定蛋白,8个细胞包被蛋白以及4个功能未知蛋白。
Acidiphilium cryptum DX1-1.细胞膜相关蛋白的表达图谱的建立,为以后各种环境中细胞膜相关蛋白的研究提供了有价值的基础性研究。从蛋白质组学角度探讨浸矿微生物铁呼吸相关的蛋白质,为微生物铁呼吸机理的研究提供一些依据。
With the reseach of iron respiration, Acidiphilium sp.has been paid more attention to,because of its iron respiration,. In the application of bioleaching
Acidiphilium sp. plays an important role during form ation of precipitated ores, which can cooperate with other microorganisms in bioleaching process.Via reducing the poisonous impact of organics to bioleaching orgnisms providing low pH enviroment, Acidiphilium sp.can effectively increase the efficiency of bioleaching.Nowadays,a microbial fuel cell has been developed using the acidophile, Acidiphilium cryptum, as the anode biocatalyst。
In this study, the cell membrane related proteins of Acidiphilium DX1-1. were extracted and separated by SDS-PAGE. And then,subjected in-gel digestion.The peptides,which were seperated by liquid chromatography,were directly subjected for online analysis by the coupled tandem mass spectrometry. With Mascot search engine,262 identification proteins were identified.
The protein export signal, subcellular location and function of each identified protein were predicted. Among these identified proteins, 13.7% possess export of the Sec type,27.86% show a twin arginine translocation (Tat) signal,18.32% have lipoprotein signal peptides.14.9% have a C-terminal GPI-anchor signal sequence,and 2.67% have both C-and N-terminal signal sequences. According to satistics assay, the molecular weights of these proteins are distributed in the range of 4KDa to 120KDa;40% of which show the pI values below 7,60% higher than 7, and 27.48% over 10.
30 identified proteins in this study are coshared between cell wall and cellmembrane enrichments, which include 3 energy metabolism related proteins,8 transport and binding proteins,2 Protein synthezing related proteins,2 modification related proteins,8 Cell envelope related proteins, and 4 proteins with unknown function.
The Acidiphilium DX1-1 membrane proteomics in this study will provide solide foundation in clarifying the mechanisms of iron respiration of Acidiphilium DX1-1 under different enviroments.
引文
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