Simple screening method for autoantigen proteins using the N-terminal biotinylated protein library produced by wheat cell-free synthesis.
Simple screening method for autoantigen proteins using the N-terminal biotinylated protein library produced by wheat cell-free synthesis.
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DOI：
10.1021/pr9010553
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发表时间：
2010-08-06
影响因子：
4.4
通讯作者：
Sawasaki, Tatsuya
中科院分区：
文献类型：
--
作者：
Matsuoka, Kazuhiro;Komori, Hiroaki;Nose, Masato;Endo, Yaeta;Sawasaki, Tatsuya
关键词：
自身免疫性疾病是一组异质性疾病，其特征在于针对主要或有限数量的身体自身抗原的免疫反应，导致炎症和组织和器官的损伤。因此，识别自身抗原是了解自身免疫性疾病的重要第一步。在这里，我们展示了一个简单的筛选方法，用于识别与患者血清抗体反应的自身抗原的N-末端生物素化蛋白质文库（BPL），使用小麦无细胞蛋白质生产系统，和市售的发光系统的组合。使用充分表征的自身抗原的优化研究表明，在均相反应条件下灵敏地检测到N-末端生物素化蛋白和抗体之间的特异性相互作用。在该优化的测定中，通过在25 μ L反应体积中加入1：500至1：10 000的稀释血清，1 μ L表达生物素化蛋白的翻译混合物产生显著的发光信号。为了构建BPL，使用214个小鼠基因（包括103个已知的自身抗原和小鼠自身免疫易感性基因座中的111个基因）和MRL/lpr小鼠的血清作为自身免疫模型。通过这种筛选方法，25个已知的自身抗原和71个蛋白质位点被确定为与血清抗体特异性反应的自身抗原蛋白。交叉引用基因本体数据库，26和38的自身抗原蛋白被预测为具有核定位，并被鉴定为膜和/或细胞外蛋白。通过免疫沉淀和/或免疫印迹分析证实了六种随机选择的蛋白质的免疫反应。有趣的是，三个自身抗原蛋白被识别的免疫沉淀，但不是通过免疫印迹分析。这些结果表明，基于BPL的方法可以提供一个简单的系统，用于筛选自身抗原蛋白质，并将有助于识别与抗体反应的自身抗原蛋白质，该抗体识别折叠的蛋白质，而不是变性或未折叠的形式。我们开发了一种新的自身抗原蛋白筛选方法相结合的N-末端生物素化的蛋白质库（BPL），使用小麦无细胞系统，和发光系统。通过这种筛选方法，将自身免疫易感位点的蛋白质鉴定为与血清抗体特异性反应的自身抗原蛋白。基于BPL的方法将有助于识别与抗体反应的自身抗原蛋白质，这些抗体识别折叠的蛋白质，而不是变性或未折叠的形式。
Autoimmune diseases are a heterogeneous group of diseases characterized by immune reactions against either a major or a limited number of the bodies own autoantigens, causing inflammation and damage to tissues and organs. Thus, identification of autoantigens is an important first step to understanding autoimmune diseases. Here we demonstrate a simple screening method for identification of autoantigens reacting with patient serum antibodies by combination of an N-terminal biotinylated protein library (BPL), produced using a wheat cell-free protein production system, and a commercially available luminescence system. Optimization studies using well-characterized autoantigens showed specific interactions between N-terminal biotinylated proteins and antibody that were sensitively detected under homogeneous reaction conditions. In this optimized assay, 1 μL of the translation mixture expressing the biotinylated proteins produced significant luminescence signal by addition of diluted serum between 1:500 and 1:10 000 in 25 μL of reaction volume. For the BPL construction, 214 mouse genes, consisting of 103 well-known autoantigens and 111 genes in the mouse autoimmune susceptibility loci, and the sera of MRL/lpr mouse were used as an autoimmune model. By this screening method, 25 well-known autoantigens and 71 proteins in the loci were identified as autoantigen proteins specifically reacting with sera antibodies. Cross-referencing with the Gene Ontology Database, 26 and 38 of autoantigen proteins were predicted to have nuclear localization and identified as membrane and/or extracellular proteins. The immune reaction of six randomly selected proteins was confirmed by immunoprecipitation and/or immunoblot analyses. Interestingly, three autoantigen proteins were recognized by immunoprecipitation but not by immunoblot analysis. These results suggest that the BPL-based method could provide a simple system for screening of autoantigen proteins and would help with identification of autoantigen proteins reacting with antibodies that recognize folded proteins, rather than denatured or unfolded forms. We developed a novel autoantigen protein screening method by combination of an N-terminal biotinylated protein library (BPL), produced using a wheat cell-free system, and a luminescence system. By this screening method, proteins in autoimmune susceptibility loci were identified as autoantigen proteins specifically reacting with sera antibodies. A BPL-based method would help identify autoantigen proteins reacting with antibodies that recognize folded proteins, rather than denatured or unfolded forms.
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影响因子：
56.9
作者：
Carninci, P;Kasukawa, T;Hayashizaki, Y
通讯作者：
Hayashizaki, Y
影响因子：
7
作者：
Beaudet, L;Bédard, J;Budarf, ML
通讯作者：
Budarf, ML
影响因子：
82.9
作者：
Robinson, WH;DiGennaro, C;Utz, PJ
通讯作者：
Utz, PJ
影响因子：
4.4
作者：
Anderson, Karen S.;Ramachandran, Niroshan;LaBaer, Joshua
通讯作者：
LaBaer, Joshua
DOI：
10.1073/pnas.87.24.9933
发表时间：
1990-12-01
影响因子：
11.1
作者：
MILLER, FW;WAITE, KA;PLOTZ, PH
通讯作者：
PLOTZ, PH