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。选择蛋白质对病毒及其结构完整性的生命周期很重要，因此代表了基于理性结构的药物设计的潜在靶标。协作组执行了实际结构确定。通过X射线衍射或多维NMR测定蛋白质的3-D结构，需要产生大量高度纯化和物理均匀的蛋白质。此外，NMR的蛋白质结构确定需要蛋白质生物合成，并与稳定同位素的组合：H-2，C-13和N-15结合。结果：（1）NEF是23 kDa蛋白对于HIV的致病性质必不可少的。我们正在研究一些涉及NEF的特定蛋白质 - 蛋白质相互作用，尤其是与CD4的39个残留细胞质尾巴相互作用。我们设计了新的表达系统，用于生产小型CD4结构域，这些结构域通常可用于产生小且相对不稳定的肽。为了研究使用NMR的相互作用，已对NEF蛋白进行了修改以增强其溶解度。这项工作仍在进行中，我们正在继续修改NEF和CD4以促进研究。 （2）HIV REV是HIV表达所需的重要调节因素。我们已经对Rev蛋白进行了广泛的修改，以提高其在NMR测量中的溶解度。 （3）艾滋病毒蛋白酶，同二聚体蛋白在病毒生命周期和主要抗AIDS药物靶标至关重要。在二聚体界面处的残基的修饰通过干扰蛋白质蛋白质缔合导致蛋白酶灭活。摘要：免疫缺陷病毒（HIV）包括许多具有调节和结构作用的蛋白质。使用重组DNA方法在细菌中表达对病毒生命周期重要的HIV蛋白，以及具有抗HIV活性的蛋白质。然后对蛋白质进行纯化，然后研究以建立其化学和物理特性。 NIH研究人员可以使用良好的蛋白质，这些研究人员研究了这些蛋白质的分子结构。这些结构信息可能为有针对性的药物设计和发现提供动力。