CORE--PROTEIN PRODUCTION AND ASSAY

核心——蛋白质生产和测定

• 批准号: 6336265
• 负责人: ELLIS L REINHERZ
• 金额: $ 17.2万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6336265
• 关键词: CD4 molecule; HIV envelope protein gp120; HIV envelope protein gp160; HIV envelope protein gp41; HeLa cells; MHC class II antigen; T lymphocyte; affinity labeling; bioassay; biomedical facility; capillary electrophoresis; cell adhesion; cell line; chemical binding; enzyme linked immunosorbent assay; human immunodeficiency virus 1; immunological substance; lymphocyte proliferation; monoclonal antibody; protein engineering; recombinant proteins; transfection; virus infection mechanism; virus replication

This core will supply large scale amount of eukaryotic and prokaryotic expression system-produced proteins. Specifically, CD4/1-183 and gp41 will be made in T7-based E. coli expression systems as inclusion bodies. In addition, Lec3.2.8.1 generated ADA and SF2 HIV-gp120 and gp160 proteins will be produced. Large scale mAb production will also be the responsibility of this core. As required, selenomethionine labeling of eukaryotically expressed protein will be undertaken for x-ray crystallography as well as /15N labeling of E. coli produced proteins for NMR analysis. Large scale mini-protein will be performed by this core once appropriate constructs have been generated. The assay component of the core will test small molecules identified by SAR by NMR as binders to CD4, gp120 or gp41 for their functional effects on CD4-gp120 interaction, HIV viral infectivity and for class II MHC interaction using a set of nine assays as detailed in the following section.

该核心将提供大量的真核生物和原核酸 表达系统生产的蛋白质。具体而言，CD4/1-183和GP41将 在基于T7的大肠杆菌表达系统中作为包容体制作。在 另外，LEC3.2.8.1生成ADA和SF2 HIV-GP120和GP160蛋白 将产生。大规模mAb生产也将是 这个核心的责任。根据需要 将用于X射线的真核生物表达的蛋白质 大肠杆菌的晶体学以及 /15N标记产生的蛋白质 NMR分析。大规模迷你蛋白将由该核心进行一次 已经生成了适当的结构。的分析部分 核心将测试由NMR鉴定为CD4的粘合剂SAR鉴定的小分子 GP120或GP41对CD4-GP120相互作用的功能作用，HIV 使用一组九个 如下部分所述的测定。