Development of the RFHR-PAGE and the Comprehensive Analysis of Nucleic Acid Binding Proteins

1. 我们开发了一种无自由基和高度还原的二维聚丙烯酰胺凝胶电泳（RFHR-PAGE）方法，用于分析核酸结合蛋白和基本蛋白，特别是核糖体蛋白。在此资助期间，对冷却系统和迁移条件等几个方面进行了改进，使其具有高度的敏感性和定量能力。目前正在与一些制造商合作，研究实际应用的可能性。在过去的两年中，从43种原核生物和9种真核生物的细胞中制备了核糖体蛋白，并通过RFHR-PAGE进行了分析。所获得的核糖体蛋白的二维斑点模式是每个物种的极具特征的。这些结果表明，二维斑点图不仅对物种的鉴定，而且对物种的进化分类都是有效的。利用RFHR-PAGE分离了大肠杆菌中存在于不溶性细胞片段（“细胞碎片”）中的数百个核酸结合蛋白片段，证实这些片段是由蛋白酶VII产生的。此外，我们在大肠杆菌核糖体中发现的一个新的核糖体蛋白C也被蛋白酶VII切割并人工转移到核糖体蛋白L31上。因此，L31这个人造物并不存在于活细胞中。蛋白质C是一种内在的核糖体蛋白，应该被称为L31，而不是旧的L31，蛋白质C的人工片段。

1. We developed the radical-free and highly reducing method of two dimensional polyacrylamide gel electrophoresis (RFHR-PAGE) for the analysis of nucleic acid binding proteins and basic proteins, in particular, ribosomal proteins. In the period of this grant, several points including the chilling system and the migration conditions were improved, resulting the highly sensitive and quantitative ability. The possibility of the practical use is being examined in cooperation with some manufactures.1. In the last two years, ribosomal proteins were prepared from cells of forty-three prokaryotes and nine eukaryotes and analyzed by the RFHR-PAGE.The obtained two dimensional spot pattern of ribosomal proteins was extremely characteristic of each species. These results showed that the two dimensional spot patterns are effective for not only the identification but also the evolutional classification of species.3. Several hundreds fragments of nucleic acid binding proteins which exist in the insoluble cell fraction ("cell debris") in E.coli were separated by the RFHR-PAGE, and it was proved that these fragments were produced by protease VII.Furthermore a new ribosomal protein C which we previously found in E.coli ribosomes was also cut by protease VII and transferred to ribosomal protein L31 artificially. Therefore L31, an artifact, does not exist in living cells. Protein C is an intrinsic ribosomal protein and should be called L31 instead of old L31, an artificial fragment of protein C.