Determination of unknown poly(ADP-ribosyl)ated proteins and binding sites

未知聚（ADP-核糖基）化蛋白和结合位点的测定

• 批准号: 16390072
• 负责人: MIWA Masanao
• 金额: $ 7.94万
• 依托单位: Nagahama Institute of Bio-Science and Technology (2005)University of Tsukuba (2004)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16390072/
• 关键词: Posttranslational modification; Poly(ADP-ribosyl)ation; Poly(ADP-ribose) glycohydrolase; Drosophila melanogaster; PARP; Centrosome; ポリADP-リボース; p53; ポリADP-リボース分解酵素; 神経変性; 細胞周期; 細胞分裂; Posttranslational modification; Poly(ADP-ribosyl)ation; Poly(ADP-ribose) glycohydrolase; Drosophila melanogaster; PARP; Centrosome; ポリADP-リボース; p53; ポリADP-リボース分解酵素; 神経変性; 細胞周期; 細胞分裂

We identified tow proteins that are poly(ADP-ribosyl)ated in vivo. They are p53 and nucleophosmin/B23. To identify the site of poly(ADP-ribosyl)ation of p53, we made the expression vector to produce GST-p53 fusion protein in E. coli. We confirmed the poly(ADP-ribosyl)ation of the entire product of p53 (aa1-aa393) by PARP-1. Currently we are preparing several GST-truncated p53 proteins, that lack about 40 amino acids, and analyzing poly(ADP-ribosyl)ated domain of p53.We identified a GST-truncated p53 protein, in which about 40 amino acids are deleted in the DNA binding domain, that lacks the acceptor activity of poly(ADP-ribosyl)ation. To identify the poly(ADP-ribosyl)ated proteins in general, we made an affinity column that have the antibody against poly(ADP-ribose), 1OH. We are optimizing the condition to bind the proteins from centrosome fractions and from other cellular fractions. To prepare a large amount of proteins that are poly(ADP-ribosylated), we are collecting the proteins from Drosophila mutant that lacks poly(ADP-ribose) glycohydrolase.

我们确定了体内聚（ADP-核糖基）的TOW蛋白。它们是p53和核素/B23。为了鉴定p53的聚（ADP-核糖基）位点，我们使表达载体在大肠杆菌中产生GST-P53融合蛋白。我们通过PARP-1证实了p53（AA1-AA393）的整个乘积的聚（ADP-核糖基）。目前，我们正在准备几种GST截断的p53蛋白，这些蛋白质缺乏约40个氨基酸，并分析了p53的聚（ADP-核糖基）ATED结构域。我们确定了一种GST折p53蛋白，其中在DNA结合域中缺乏大约40个氨基酸的p53蛋白。为了确定一般的聚（ADP-核糖基）atted蛋白，我们制作了一个亲和力柱，具有抗聚（ADP-核糖）1OH的抗体。我们正在优化结合中心体馏分和其他细胞级分的蛋白质的条件。为了制备大量的聚（ADP-核糖基化）的蛋白质，我们正在从果蝇突变体中收集缺少聚（ADP-核糖）糖含量糖酶的蛋白质。