Functional Analysis of Proteins regulated by Ubiquitin Ligase

我们试图通过建立检测基因工程前后差异的综合分析方法来鉴定泛素连接酶的生理底物。首先，我们通过抗泛素抗体特异性地纯化泛素蛋白。泛素是一种丰富的蛋白质，在哺乳动物细胞中以自由形式(非结合或结合形式)存在。用常规抗体亲和层析，主要回收游离泛素，不能收集泛素结合蛋白。为了提高收集效率，我们对多种抗体进行了检测，最终决定使用FK2单抗，它只与底物结合的泛素特异结合。我们利用FK2结合柱亲和层析成功地浓缩了泛素化蛋白，为了比较基因敲除小鼠和野生型小鼠的蛋白质，必须选择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，在此基础上制备了Fbw7基因缺陷的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了全细胞裂解液。细胞裂解物负载在柱填充FK2-抗体结合珠上，用于泛素化蛋白质的浓缩。我们计划比较Fbw7缺陷的胚胎成纤维细胞和野生型胚胎成纤维细胞泛素化蛋白的差异。

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.