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Functional Analysis of Proteins regulated by Ubiquitin Ligase

泛素连接酶调控的蛋白质的功能分析

基本信息

批准号：
16370084
负责人：
NAKAYAMA Keiko
金额：
$ 9.86万
依托单位：
Tohoku University
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

项目摘要

We have tried to identify physiological substrates of ubiquitin ligase by establishment of comprehensive-analytical method for detection of difference between before and after genetic engineering in knock-out mice.First, we purified ubiquitinated protein specifically by anti-ubiquitin antibody. Ubiquitin is an abundant protein and exist as free form (not conjugated or binding form in mammalian cells. By affinity-chromatography using a conventional antibody, we recovered a free-ubiqitin mainly, and could not collect ubiquitin conjugated proteins. For improvement of the efficiency of collection, we assesed many kinds of antibodies and finally decided to use the FK2 monoclonal antibody, which binds only substrate-conjugated ubiqitin specifically. We succeeded in concentrating ubiquitinated proteins by affinity-chromatography using FK2 binding column.For comparison of proteins between knock-out mice and wild type mice, we have to choose the most adequate tissue and the most effective method for cell-extraction. We identified the tissue which expressed F-box protein we were interested in by in-situ hybridization.Based on these basic informations, we prepared Fbw7-deficient-embryonic fibroblasts and also wild-type fibroblast, and extracted whole cell lysate from each samples. Cell lysate loaded on column packed FK2-antibody conjugated beads for concentration of ubiquitinated proteins. We plan to compare the difference of the ubiquitinated proteins between Fbw7-deficient and wild-type embryonic fibroblasts.

本研究通过建立基因工程敲除小鼠泛素连接酶生理底物的综合分析方法，检测基因工程前后泛素连接酶的差异。泛素是一种丰富的蛋白质，在哺乳动物细胞中以游离形式（非缀合或结合形式）存在。通过使用常规抗体的亲和层析，我们主要回收游离泛素，并且不能收集泛素缀合的蛋白质。为了提高收集效率，我们评估了多种抗体，最终决定使用仅特异性结合底物缀合的泛素的FK 2单克隆抗体。为了比较基因敲除小鼠和野生型小鼠的蛋白质，我们选择了最合适的组织和最有效的细胞提取方法。我们通过原位杂交鉴定了表达我们感兴趣的F-box蛋白的组织，基于这些基本信息，我们制备了Fbw 7缺陷型胚胎成纤维细胞和野生型成纤维细胞，并从每个样品中提取全细胞裂解物。将细胞裂解物加载到柱填充的FK 2-抗体缀合珠上，用于浓缩泛素化蛋白。我们计划比较Fbw 7缺陷型和野生型胚胎成纤维细胞之间泛素化蛋白的差异。