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用免疫亲和层析法纯化萝卜 PHGPx 天然蛋白
引用本文:杨晓东,刘进元.用免疫亲和层析法纯化萝卜 PHGPx 天然蛋白[J].生物化学与生物物理进展,2005,32(8):794-799.
作者姓名:杨晓东  刘进元
作者单位:清华大学生物科学与技术系,分子生物学实验室及教育部蛋白质科学重点实验室,北京,100084
基金项目:国家自然科学基金资助项目(30170080,39770078)和国家重点基础研究发展规划资助项目(973)(2004CB117300).
摘    要:萝卜磷脂氢谷胱甘肽过氧化物酶 (RsPHGPx) 是一个定位于线粒体的蛋白质 . 为了阐明该蛋白质线粒体定位信号的准确切割位点,采用了免疫亲和层析方法纯化天然的 RsPHGPx. 用重组 RsPHGPx 蛋白免疫兔子获得了抗 RsPHGPx 的多克隆抗血清,以重组 RsPHGPx 蛋白为配体,采用亲和层析技术对抗血清进行了纯化,得到了单特异性的抗 RsPHGPx 的抗体 . 将纯化好的抗体偶联到一个 N- 羟基琥珀酰亚胺 (NHS) 预先激活的琼脂糖柱子上,装配成一个以单特异性的抗 RsPHGPx 抗体为配体的免疫亲和层析柱 . 经过对纯化条件的摸索和优化,形成了一个简单、特异的一步法纯化方案 . 按照该方案,从萝卜幼苗线粒体总蛋白质提取物中纯化到一个分子质量与预期值相一致的特异蛋白质 . 免疫印迹分析表明,该蛋白质被抗 RsPHGPx 的抗血清特异识别 . 酶活性分析表明,该蛋白质具有显著的 PHGPx 活性 . 这些结果表明,纯化到的特异蛋白质是萝卜的 RsPHGPx 天然蛋白 . 这是首个关于定位于植物细胞器的 PHGPx 蛋白纯化的报道 . 这一结果为准确测定 RsPHGPx 信号肽的切割位点奠定了基础,并将有助于对植物 PHGPx 的亚细胞定位机制及其生理功能的深入研究 .

关 键 词:免疫亲和层析,天然蛋白质,多克隆抗体,磷脂氢谷胱甘肽过氧化物酶,萝卜
收稿时间:03 9 2005 12:00AM
修稿时间:2005-03-092005-03-28

Micropreparation of a Native PHGPx Protein From Radish Seedlings by Immunoaffinity Chromatography
YANG Xiao-Dong and LIU Jin-Yuan.Micropreparation of a Native PHGPx Protein From Radish Seedlings by Immunoaffinity Chromatography[J].Progress In Biochemistry and Biophysics,2005,32(8):794-799.
Authors:YANG Xiao-Dong and LIU Jin-Yuan
Affiliation:Laboratory of Molecular Biology and Protein Science Laboratory of The Ministry of Education, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China;Laboratory of Molecular Biology and Protein Science Laboratory of The Ministry of Education, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China
Abstract:Radish phospholipid hydroperoxide glutathione peroxidase (RsPHGPx) was identified as a mitochondrion-targeting PHGPx in previous work. To determine its cleavage site of the targeting peptide, the immunoaffinity chromatography (IAC) purification approach was carried out to isolate the native RsPHGPx protein. Polyclonal antibodies directed against recombinant RsPHGPx were raised in rabbit. Monospecific anti-RsPHGPx antibodies were isolated by means of affinity chromatography using the recombinant RsPHGPx as affinity ligand, and employed in assembling an IAC column. A single-step, highly specific and easy-to-use protocol was developed for purification of the active RsPHGPx protein through the assembled IAC column. Using this approach, a specific protein of the expected molecular size was obtained from the mitochondrial fraction of radish seedlings. Western blot analysis showed that it could be specifically recognized by anti-RsPHGPx antibodies, and an enzyme activity assay indicated that it exhibited significant PHGPx activity, suggesting that the purified protein should be the desired native RsPHGPx. These results will lead to clarification of the targeting peptide and the active mature protein of RsPHGPx and will be helpful to further probe the intracellular localization mechanism and biological function of this plant PHGPx.
Keywords:immunoaffinity chromatography  native protein  polyclonal antibodies  phospholipid hydroperoxide glutathione peroxidase  radish
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