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

我们在体内鉴定了两种聚（adp -核糖基）蛋白。它们是p53和核磷蛋白/B23。为了确定p53的聚（adp -核糖基）化位点，我们在大肠杆菌中制作了生成GST-p53融合蛋白的表达载体。我们用PARP-1证实了整个p53产物（aa1-aa393）的聚（adp -核糖基）化。目前，我们正在制备几种gst截断的p53蛋白，这些蛋白缺少约40个氨基酸，并分析p53的聚（adp -核糖基）化结构域。我们发现了一个gst截断的p53蛋白，其中DNA结合域缺失了大约40个氨基酸，缺乏聚（adp -核糖基）化的受体活性。为了从总体上鉴定聚（adp -核糖）蛋白，我们制作了一个具有抗聚（adp -核糖）抗体1OH的亲和柱。我们正在优化条件以结合中心体和其他细胞片段的蛋白质。为了制备大量的聚（adp -核糖基化）蛋白，我们从缺乏聚（adp -核糖）糖水解酶的果蝇突变体中收集蛋白质。

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.