ISOLATION/PURIFICATION MAMMALIAN PROSTACYCLIN SYNTHASE

分离/纯化哺乳动物前列环素合成酶

• 批准号: 3508703
• 负责人: ROBERT G HARDS
• 金额: $ 25.66万
• 依托单位: OXFORD BIOMEDICAL RESEARCH, INC.
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-30 至 1993-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3508703
• 关键词: Escherichia coli; SDS polyacrylamide gel electrophoresis; affinity chromatography; animal tissue; antibody; antibody specificity; cardiovascular disorder chemotherapy; complementary DNA; drug design /synthesis /production; electrofocusing; electroporation; enzyme mechanism; enzyme substrate analog; fatty acid metabolism; fatty acid synthase; gel filtration chromatography; gene expression; genetic library; hydropathy; immunoperoxidase; ion exchange chromatography; molecular cloning; monoclonal antibody; nucleic acid probes; prostacyclins; protein purification

Prostacyclin synthase catalyzes an intramolecular redox reaction in which prostaglandin endoperoxide, PGH2, is converted to prostacyclin. The potent biological activities of prostacyclin indicate an important role for prostacyclin synthase in the pathophysiology of thrombosis and cardiovascular diseases. Commercial availability of a purified preparation of mammalian prostacyclin synthase should therefore facilitate basic and pharmaceutical research, including studies of factors involved in the regulation of prostacyclin synthase gene expression, development of novel therapeutic agents for the treatment of cardiovascular diseases, and development of new strategies for the prevention and management of thromboembolic episodes. In Phase II, we will purify prostacyclin synthase from ram seminal vesicles to homogeneity using a variety of methodologies including ion- exchange chromatography, affinity chromatography, isoelectric focusing and gel permeation chromatography. The purified protein will be used to raise both poly- and monoclonal antibodies. The polyclonal antibodies will be used in conjunction with a lambda-gt11 expression library to isolate cDNA sequences specific for prostacyclin synthase. These cDNA sequences will then be used to produce DNA and RNA probes. Attempts will be made to use the monoclonal antibodies developed in a large scale, single step purification procedure. The purified enzyme, the antibodies and the nucleic acid probes will be marketed Oxford Biomedical Research.

前列环素合成酶催化分子内氧化还原反应 前列腺素内源性过氧化产物(PGH2)被转化为前列腺素。这个 前列环素的强大生物活性表明它在 前列环素合成酶在血栓形成的病理生理学中的作用 心血管疾病。一种纯净品的商业供应 因此，哺乳动物前列环素合成酶的制备 促进基础和药物研究，包括研究 前列环素合酶基因调控的相关因素 治疗骨肉瘤的新型治疗药物的表达和开发 心血管疾病，以及开发新的治疗策略 血栓栓塞症的预防和处理。 在第二阶段，我们将从公羊精液中纯化前列环素合成酶。 使用各种方法将囊泡转化为均一性，包括离子- 交换层析、亲和层析、等电聚焦 凝胶渗透层析法。纯化的蛋白质将用于 同时提高多克隆抗体和单抗的水平。多克隆抗体 将与lambda-gt11表达式库结合使用 分离前列环素合成酶特异的c DNA序列。这些 然后，cDNA序列将被用于生产DNA和RNA探针。 将尝试使用在一种 规模化、一步提纯工艺。纯化的酶， 抗体和核酸探针将在牛津大学上市 生物医学研究。