DETERMINATION OF UNKNOWN POLY(ADP-RIBOSYL)ATED PROTEINS AND BINDING SITES

未知聚（ADP-核糖基）化蛋白质和结合位点的测定

基本信息

批准号：
18590277
负责人：
MIWA Masanao
金额：
$ 2.57万
依托单位：
Nagahama Institute of Bio-Science and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

To understand the physiological function of polyADP-ribosylation, it is absolutely necessary to identify the acceptor proteins of polyADP-ribosylation. We prepared and quantities of monoclonal antibody against poly(ADP-ribose) and purified it for affinity chromatography. During the course of experiments, we noticed that the extracted polyADP-ribosylated proteins did not bind to the affinity column suggesting that there are other proteins binding to poly(ADP-ribose) that inhibit the binding of polyADP-ribosylated proteins to the column. To solve these problems, we used human kidney cell line (293T) as cell lysate and synthesized polyADP-ribosylated proteins in vitro and checked the ideal condition of extraction of polyADP-ribosylated proteins. It turned out that extraction buffer should contain 0.1% SDS for better extraction. For extracting the polyADP-ribosylated proteins from Drosophila mutant that lacks poly(ADP-ribose) glycohydrolase and have accumulated polyADP-ribosylated proteins in vivo, good extraction efficiency was obtained when the buffer contains 0.5% or more SDS. However it became also evident that polyADP-ribosylated proteins do not bind to the antibody column, presumably SDS did inhibit the binding. We had some success in eliminating the free SDS by adding the nonionic detergent in our preliminary experiments. Thus we are now trying to purify the polyADP-ribosylated proteins.

为了了解聚ADP-核糖基化的生理功能，绝对有必要鉴定聚ADP-核糖基化的受体蛋白。我们制备了大量的抗聚（ADP-核糖）单克隆抗体，并将其纯化用于亲和层析。在实验过程中，我们注意到提取的聚ADP-核糖基化蛋白没有与亲和柱结合，这表明还有其他蛋白质与聚（ADP-核糖）结合，抑制了聚ADP-核糖基化蛋白与亲和柱的结合。为了解决这些问题，我们使用人肾细胞系（293T）作为细胞裂解液，在体外合成了聚ADP-核糖基化蛋白，并检查了聚ADP-核糖基化蛋白的理想提取条件。结果表明，提取缓冲液应含有 0.1% SDS，以便更好地提取。为了从缺乏聚(ADP-核糖)糖水解酶且体内积累了聚ADP-核糖化蛋白的果蝇突变体中提取聚ADP-核糖化蛋白，当缓冲液含有0.5%或更多SDS时获得良好的提取效率。然而，也很明显，聚 ADP 核糖基化蛋白不与抗体柱结合，推测 SDS 确实抑制了这种结合。在我们的初步实验中，通过添加非离子洗涤剂，我们在消除游离 SDS 方面取得了一些成功。因此，我们现在正尝试纯化聚ADP核糖基化蛋白质。

项目成果

期刊论文数量（0）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Induction of centrosome amplification by inhibitors of poly(ADP-ribose)polymerases

聚（ADP-核糖）聚合酶抑制剂诱导中心体扩增

DOI：
发表时间：
2007
期刊：
影响因子：
0
作者：
Kikuchi H;Ozaki T;Furuya K;Hanamoto T;Nakanishi M;Yamamoto H;Yoshida K;Todo S;Nakagawara A.;Masanao Miwa
通讯作者：
Masanao Miwa

PolyADP-ribosylation and cancer