Production Of HIV And HIV Related Proteins For Structura

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)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和结晶试验中的溶解度。此外，我们正试图通过与抗体和微管蛋白形成二元配合物来进一步稳定Rev，以便进行结构研究。(2) Nef是一个23 kDa的蛋白质，对HIV的致病特性至关重要。我们正在继续研究一些涉及Nef的特定蛋白质-蛋白质相互作用，特别是涉及HIV-1反激活蛋白Tat。我们已经使用表面等离子体共振来显示这些蛋白质之间的特异性和紧密结合。利用Nef和Tat的蛋白质工程变体，我们正在制备复合物以进行更详细的结构研究(3)HIV蛋白酶，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。含硫氨基酸（半胱氨酸或蛋氨酸）在二聚体界面的氧化通过干扰蛋白质-蛋白质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。

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) 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 and crystallization trials. In addition, we are attempting to further stabilize Rev for structural studies by forming binary complexes with antibodies and tubulin. (2) Nef is a 23 kDa protein essential for the pathogenic properties of HIV. We are continuing to investigate some of the specific protein-protein interactions involving Nef especially involving the HIV-1 transactivating protein Tat. We have used surface plasmon resonance to show a specific and tight binding between these proteins. Using protein engineered variants of Nef and Tat we are preparing complexes for more detailed structural study (3) HIV protease, a homodimeric protein is essential in the viral life cycle and a major anti-AIDS drug target. The oxidation of sulphur containing amino acids (cysteine or methionine) 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.