NOVEL SYSTEM FOR REGULATED HIGH LEVEL GENE EXPRESSION

调控高水平基因表达的新系统

• 批准号: 3507981
• 负责人: THOMAS R FUERST
• 金额: $ 20.67万
• 依托单位: MEDIMMUNE, INC.
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-27 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3507981
• 关键词: DNA directed RNA polymerase; Escherichia coli; Mammalia; bioreactors; biotechnology; chimeric proteins; fusion gene; gene expression; gene induction /repression; genetic regulation; genetic regulatory element; genetic techniques; genetic translation; lac operon; messenger RNA; method development; molecular cloning; mutant; posttranslational modifications; protein biosynthesis; protein folding; protein transport; recombinant DNA; tissue /cell culture; vaccinia virus

DESCRIPTION (Adapted from applicant's abstract): Numerous expression systems exist for the bioproduction of proteins in both prokaryotic and eukaryotic cells. Each system has its own set of advantages as well as shortcomings. The need remains for a mammalian cell expression system that can achieve high-level gene expression, correctly process proteins of mammalian origin, and is economical and easy to use. The aim is of this proposal is to establish a mammalian cell expression system capable of rapid and high level production of specified proteins. To this end, the use of regulatory components from the E. coli lactose operon to regulate the expression of bacteriophage T7 RNA polymerase in a single vaccinia virus-based vector system was investigated. The requirement to control T7 RNA polymerase expression was necessary since previous attempts to develop such a single virus system without regulation were unsuccessful, presumably due to the high transcriptase activity and processibility of the phage RNA polymerase causing interference with endogenous viral transcription or replication. In Phase I, a single recombinant vaccinia virus harboring both T7 RNA polymerase and T7 promoter-controlled target gene sequences under regulatory control of genetic elements from the lactose operon was successfully constructed. Induction of this system led to a higher level of target gene expression than that obtained previously using a two virus system. In Phase II, the expression parameters for this system will be further optimized. This system will be adapted to pilot-level bioreactor instrumentation leading to the bioproduction of several proteins of interest. New strategies to further refine and expand the utility of this system will be evaluated for the commercial production of proteins.

描述（改编自申请人的摘要）：许多表达 存在用于蛋白质和蛋白质生物生产的系统 真核细胞。 每个系统都有自己的一套优势以及 缺点。 仍然需要哺乳动物细胞表达系统 可以实现高级基因表达，正确处理的蛋白质 哺乳动物起源，是经济且易于使用的。 目的是 建议是建立能够的哺乳动物细胞表达系统 指定蛋白质的快速生产。 为此， 使用大肠杆菌乳糖操纵子的调节组件来调节 噬菌体T7 RNA聚合酶在单个离子酵母中的表达 研究了基于病毒的媒介系统。 控制T7的要求 由于先前尝试开发的RNA聚合酶表达是必要的 这样的单一病毒系统没有调节，可能是不成功的，大概是 由于转录酶的活性高和噬菌体RNA的过程 聚合酶导致干扰内源性病毒转录或 复制。 在第一阶段，携带的单个重组离子病毒 T7 RNA聚合酶和T7启动子控制的靶基因序列 在监管乳糖操纵子的遗传元素的调节下 成功构建。 该系统的诱导导致更高级别 靶基因表达的表达比先前使用两个病毒获得的表达 系统。 在第二阶段，该系统的表达参数将是 进一步优化。 该系统将适用于飞行员级生物反应器 仪器导致几种蛋白质生物生产 兴趣。 进一步完善和扩展实用性的新策略 系统将评估蛋白质的商业生产。