AN RNA POLYMERASE FOR SYNTHESIS OF CAPPED RNAS

用于合成加帽 RNA 的 RNA 聚合酶

• 批准号: 6134241
• 负责人: Linda A. Guarino
• 金额: $ 10万
• 依托单位: EPICENTRE TECHNOLOGIES CORPORATION
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6134241
• 关键词: Baculoviridae; DNA directed RNA polymerase; RNA biosynthesis; bacterial proteins; biotechnology; genetic transcription; polyadenylate; posttranscriptional RNA processing; protein purification; technology /technique development

Baculovirus Expression Vectors are widely used for high-level protein production with applications in medical therapeutics, diagnostics, vaccines, and drug discovery. The power of the baculovirns system is due to the viral RNA polymerase. This is a simple four-subunit complex that has, in addition to its catalytic activity, the ability to specifically recognize viral promoters and to process RNAs at both the 5' and 3' ends. The goal of this proposal is to develop baculovirus RNA polymerase as a commercial tool for the production of capped and polyadenylated transcripts. There are no commercially available enzymes for simultaneous transcription and processing of RNAs in vitro. Several companies sell kits for the production of capped RNAs. but this requires the use of cap analog. Baculovirus RNA polymerase should produce capped RNAs more cheaply and efficiently, and would also polyadenylate the RNAs which should provide increased stability and efficiency of translation. Our specific aims are to: 1) Develop a bacterial expression system for baculovirus RNA polymerase; 2) Design a purification scheme that yields enzyme that is 95% pure and 95% active; and 3) Analyze the promoter specificity of baculovirus RNA polymerase. In Phase II of the program, we will develop commercial products, including an improved coupled transcription-translation system and a diagnostic kit for protein truncation assays. PROPOSED COMMERCIAL APPLICATION: Baculovirus RNA polymerase will be developed as a commercial tool for the production of capped and polyadenylated transcripts. This will be useful for researchers needing RNAs with increased stability and improved translational efficiency in vivo and in vitro. In addition, baculovirns RNA polymerase should offer improved thoughput for medical diagnostic assays based on coupled transcription/translation systems like the protein truncation test.

杆状病毒表达载体被广泛用于高级蛋白质生产，并在医学治疗，诊断，疫苗和药物发现中应用。 Baculovirns系统的功率是由于病毒RNA聚合酶。这是一个简单的四元配合物，除了它的催化活性外，还具有专门识别病毒启动子并在5'和3'末端处理RNA的能力。该提案的目的是开发杆状病毒RNA聚合酶作为生产限制和聚腺苷酸化转录本的商业工具。在体外RNA的同时转录和加工没有市售的酶。几家公司出售工具包以生产限制RNA。但这需要使用帽类似物。杆状病毒RNA聚合酶应更便宜，有效地产生限制的RNA，并且还将聚腺苷酸化RNA，这应该提供提高的稳定性和翻译效率。我们的具体目的是：1）开发杆状病毒RNA聚合酶的细菌表达系统； 2）设计一种纯化方案，可产生95％纯度和95％活跃的酶； 3）分析杆状病毒RNA聚合酶的启动子特异性。在该计划的第二阶段中，我们将开发商业产品，包括改进的耦合转录 - 翻译系统和用于蛋白质截断测定法的诊断试剂盒。拟议的商业应用：杆状病毒RNA聚合酶将作为生产限制和聚腺苷酸化转录本的商业工具开发。这对于需要RNA具有提高稳定性并提高体内和体外翻译效率的研究人员很有用。此外，基于蛋白质截断测试（例如蛋白质截断测试），Baculovirns RNA聚合酶应为基于转录/翻译系统（例如转录/翻译系统）提供改进的医学诊断测定法。