EXPRESSION AND SCALED-UP PREPARATION OF THE HIV INTEGRASE

逆转录病毒生命周期中的一个重要步骤是DNA的整合 复制到宿主基因组中 这种整合是由 病毒编码的整合酶，IN。 IN蛋白是一个有吸引力的靶点 对于抗逆转录病毒药物治疗，因为IN是病毒感染所必需的， 传播，因为IN同源物可能不起重要作用， 宿主细胞的健康或存活。 抗IN药物的合理设计 这取决于对IN结构的了解。 的总目标 这个项目是为了促进解决的X射线晶体结构的 由HIV逆转录病毒编码的IN蛋白， 获得性免疫缺陷综合症（艾滋病）。 我们相信，一个专注的， 集中精力使可溶形式的IN的产量最大化， 证明是促进其结晶学的最有效方法。 因为即使是氨基酸序列中微小的变化 对蛋白质的溶解度和一个人的能力产生深远的影响， 结晶，由HIV-1和HIV-2编码的IN蛋白将被 研究了 天然IN的过量生产和溶解度将是 使用噬菌体T7表达系统进行了系统研究。 在 此外，我们将构建表达系统的反向 转录酶-IN融合蛋白，IN从该融合蛋白中被蛋白水解切割， 体内，以及对于与麦芽糖结合蛋白（MBP）的融合蛋白， 谷胱甘肽-S-转移酶（GST），这两者都被发现可以增强 外源蛋白在E.杆菌 表达 水平、产品溶解度和酶活性将被表征。 IN的部分蛋白水解将用于定义蛋白质结构域，稳定 蛋白水解片段将被表征， 将被建造。

An essential step in the retroviral life cycle is the integration of a DNA copy of the virus into the host genome. This integration is catalyzed by the virus-encoded integrase, IN. The IN protein is an attractive target for anti-retroviral drug therapy both because IN is essential for viral propagation and because IN homologs probably play no role important to the health or survival of the host cell. The rational design of anti-IN drugs depends upon the knowledge of the structure of IN. The overall goal of this project is to facilitate solution of the X-ray crystal structure of the IN protein encoded by the HIV retrovirus, the etiological agent of Acquired Immunodeficiency Syndrome (AIDS). We believe that a focussed, concentrated effort to maximize production of soluble forms of IN will prove to be the most efficient way to facilitate its crystallography. Because even small and subtle changes in amino acid sequence can have profound effects on the solubility of a protein and on one's ability to crystalize it, the IN proteins encoded by both HIV-1 and HIV-2 will be investigated. Overproduction and solubility of native IN will be systematically studied using the bacteriophage T7 expression system. In addition we will construct expression systems for the reverse transcriptase-IN fusion protein from which IN is proteolytically cleaved in vivo, and for fusion proteins with the maltose binding protein (MBP) and glutathione-S-transferase (GST) both of which have been found to enhance solubility of foreign proteins overexpressed in E. coli. Expression levels, product solubility and enzymatic activities will be characterized. Partial proteolysis of IN will be used to define protein domains, stable proteolytic fragments will be characterized and genes for their expression will be constructed.