Production Of HIV And HIV Related Proteins For Structura

用于 Structura 的 HIV 和 HIV 相关蛋白的生产

• 批准号: 6680165
• 负责人: PAUL T WINGFIELD
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6680165
• 关键词: HIV envelope protein; X ray crystallography; antiAIDS agent; antineoplastic antibiotics; biotechnology; conformation; drug design /synthesis /production; endopeptidases; human immunodeficiency virus; integrase; nuclear magnetic resonance spectroscopy; plant proteins; protein biosynthesis; protein engineering; protein purification; protein structure function; recombinant proteins; stable isotope; structural biology; virus protein

Background: HIV and HIV-related proteins are produced by recombinant DNA methods for high-resolution structural analyses. The proteins are selected which are important for the life cycle of the virus and for its structural integrity and, thus, represent potential targets for rational structure-based drug design. Collaborating groups performed the actual structure determinations. The determination of the 3-D structure of proteins by X-ray diffraction or multidimensional NMR required the production of large quantities of highly purified and physically homogeneous protein. In addition, protein structure determination by NMR requires protein biosynthetically labeled with combinations of the stable isotopes: H-2, C-13 and N-15. Results: (1) Nef is a 23 kDa protein essential for the pathogenic properties of HIV. We are investigating some of the specific protein-protein interactions involving Nef especially interaction with the 39-residue cytoplasmic tail of CD4. We have devised novel expression systems for producing the small CD4 domain that are useful in general for producing small and relatively unstable peptides. To study the interaction using NMR, the Nef protein has been modified to enhance its solubility. This work is still in progress and we are continuing to modify both Nef and CD4 to facilitate the study. (2) HIV Rev is an important regulatory factor required for HIV expression. We have made extensive modifications of the Rev protein in order to improve its solubility for NMR measurements. (3) HIV protease, a homodimeric protein is essential in the viral life cycle and a major anti-AIDS drug target. The modification of residues at the dimer interface result in protease inactivation by interfering with protein-protein association. Summary: the immunodeficiency virus (HIV) comprises a number of proteins with regulatory and structural roles. HIV proteins important for the virus life cycle, and proteins which have anti-HIV activity, are expressed in bacteria using recombinant DNA methods. The proteins are purified then studied to establish their chemical and physical properties. Well-characterized proteins are made available to NIH investigators who study the molecular structure of these proteins. This structural information may provide impetus for targeted drug design and discovery.

背景：HIV和HIV相关蛋白是通过重组DNA方法产生的，用于高分辨率结构分析。所选择的蛋白质对病毒的生命周期及其结构完整性很重要，因此代表了合理的基于结构的药物设计的潜在靶标。协作小组执行实际的结构确定。通过x射线衍射或多维核磁共振测定蛋白质的三维结构需要生产大量高度纯化和物理均匀的蛋白质。此外，通过核磁共振测定蛋白质结构需要用稳定同位素H-2、C-13和N-15组合进行生物合成标记。结果：(1)Nef是一个23 kDa的蛋白，对HIV的致病特性至关重要。我们正在研究一些涉及Nef的特异性蛋白-蛋白相互作用，特别是与CD4细胞的39个残基细胞质尾部的相互作用。我们设计了新的表达系统来产生小的CD4结构域，这对于产生小的和相对不稳定的肽是有用的。为了利用核磁共振研究相互作用，对Nef蛋白进行了修饰以提高其溶解度。这项工作仍在进行中，我们将继续修改Nef和CD4以促进研究。(2) HIV Rev是HIV表达所需的重要调控因子。我们对Rev蛋白进行了广泛的修饰，以提高其在核磁共振测量中的溶解度。(3) HIV蛋白酶是一种同型二聚体蛋白，在病毒生命周期中是必不可少的，也是抗艾滋病药物的主要靶点。二聚体界面残基的修饰通过干扰蛋白-蛋白结合导致蛋白酶失活。摘要：免疫缺陷病毒（HIV）由许多具有调节和结构作用的蛋白质组成。利用重组DNA方法在细菌中表达对病毒生命周期重要的HIV蛋白和具有抗HIV活性的蛋白质。这些蛋白质被纯化，然后被研究以确定它们的化学和物理性质。表征良好的蛋白质提供给研究这些蛋白质分子结构的NIH研究人员。这种结构信息可能为靶向药物的设计和发现提供动力。