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VIRAL DISPLAY OF MEMBRANE PROTEINS AND ITS APPLICATION TO PROTEIN INTERACTION ANALYSIS

膜蛋白的病毒展示及其在蛋白质相互作用分析中的应用

基本信息

批准号：
13558084
负责人：
HAMAKUBO Takao
金额：
$ 9.02万
依托单位：
THE UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

Membrane proteins are the major targets for the genome based drug discovery in the post-genomic era. In this research we developed a novel expression system for membrane proteins using baculovirus and utilized it to raise specific antibodies against membrane proteins and nuclear receptor superfamily. We found that a novel property of baculovirus expression system that some membrane proteins can be displayed on the budded virus itself. To date we have observed the membrane display of several ER proteins such as sterol regulatory element-binding proteirt-2 (SREBP-2), SREBP cleavage-activating protein (SCAP), HMG-CoA reductase, presenilin, and nicastrin. All of them had proper activities. Also we have succeeded to reconstitute the trimeric G proteins and G protein coupled receptors on the budded baculovirus. Those reconstituted GPCR had high affinity for ligand binding. Thus this viral expression system considered to be very useful for the analysis of membrane proteins. We then utilized this system for making antibody against membrane proteins. We have been succeeded to generate specific monoclonal antibodies against several membrane proteins such as transporter, SCAP, and nicastrin. On the other hand, using gp64 fusion protein, a membrane protein of baculovirus itself, series of monoclonal antibodies has been generated against superfamily of nuclear receptor. We have been achieved to raise 30 monoclonal antibodies out of 48 nuclear receptor family proteins using this system. Thus we have been developed novel viral display system for membrane proteins and its application for antibody generation.

膜蛋白是后基因组时代基于基因组的药物发现的主要靶点。本研究利用杆状病毒构建了一种新型的膜蛋白表达系统，并利用该系统制备了针对膜蛋白和核受体超家族的特异性抗体。我们发现杆状病毒表达系统的一个新特性是某些膜蛋白可以在出芽病毒上展示。到目前为止，我们已经观察到几种ER蛋白的膜展示，如固醇调节元件结合蛋白2（SREBP-2），SREBP裂解激活蛋白（SCAP），HMG-CoA还原酶，早老素和nicastrin。他们都有适当的活动。我们还成功地重组了出芽杆状病毒上的三聚体G蛋白和G蛋白偶联受体。这些重组GPCR具有高亲和力的配体结合。因此，这种病毒表达系统被认为是非常有用的膜蛋白的分析。然后，我们利用这个系统来制造针对膜蛋白的抗体。我们已经成功地产生了针对几种膜蛋白，如转运蛋白，SCAP和nicastrin的特异性单克隆抗体。另一方面，利用杆状病毒自身的膜蛋白gp 64融合蛋白，制备了一系列抗杆状病毒核受体超家族的单克隆抗体。利用该系统，我们已经成功地从48个核受体家族蛋白中制备了30个单克隆抗体。因此，我们已经开发了新的膜蛋白的病毒展示系统及其在抗体产生中的应用。