Large-scale Production & Purification Of Compounds With

大规模生产

• 批准号: 6503224
• 负责人: Joseph Shiloach
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6503224
• 关键词: Clostridium; Escherichia coli; HeLa cells; Salmonella typhi; Shigella vaccines; bacterial proteins; biological products; biotechnology; chemical registry /resource; diphtheria toxin; fermentation; glucose metabolism; immunological substance; method development; microarray technology; microorganism mass culture; microorganism resource /registry; protein purification; recombinant DNA; recombinant proteins; tissue /cell culture; vaccine development; vaccinia virus

The Biotechnology Unit is responsible for the production and purification of biological material. The Unit undertakes a wide array of biological production tasks such as large-scale growth of prokaryotes and eucaryotes, and production and purification of biological compounds especially proteins. These products, not available from commercial sources, are needed for clinical and structural studies. The Biotechnology Unit is capable of handling these various production tasks by implementing both physiological and technical approaches. Efficient process development and production is possible since the Unit possesses two integrated elements: a fully operational multi-purpose pilot production facility and the capability of conducting research and process development. During the last year the unit performed approximately 200 different, large scale preparations including: 1) growing various microorganisms such as recombinant Escherichia coli, recombinant Saccharomyces cerevisiae, recombinant Pichia pastoris, mutant strains of Corynebacteria diphtheria, several types of Salmonella and Shigella and mutant strain of Bacillus anthracis; 2) propagating large volumes of various eucaryotic cells such as HeLa, CHO, drosophila and Sf-9; 3) purifying gram quantities of diphtheria toxin CRM (Cross Reacting Material) and recombinant Pseudomonas aeruginosa exotoxin A needed for the preparation of conjugated vaccines. In the research and development arena, the unit continues its work in the following topics: 1) development of more efficient methods for large scale protein recovery, here we performed experiments implementing expanded bed adsorption techniques for capturing proteins on ion exchangers, chelating and hydrophobic resins; 2) conducting research aimed at understanding the glucose metabolism of various recombinant E. coli strains in an effort to find out why certain strains are more suitable for foreign protein production using microarrays technology and protein analysis; 3) developing fermentation procedures for expressing sensitive, recombinant clostridium protein from e.coli by suppressing proteolytic activity; and 4) conducting experiments for improving the production protocol of recombinant proteins from Pichia pastoris, especially endostatin. In addition, the unit continues its research work in two topics, one is on the effect of various high density growth strategies of recombinant E.coli, on the production of recombinant proteins, and the other is on the production process of recombinant proteins from the HeLa/vaccinia system. In collaboration with LDMI, NICHD, the unit conducted work on the development of new vaccine against Salmonella typhi, and shigella flexneri. The products are currently in field trials.

生物技术股负责生物材料的生产和提纯。该单位承担着广泛的生物生产任务，如原核生物和真核生物的大规模生长，以及生物化合物特别是蛋白质的生产和纯化。这些产品无法从商业来源获得，需要用于临床和结构研究。生物技术股能够通过实施生理和技术方法来处理这些不同的生产任务。高效率的工艺开发和生产是可能的，因为该股拥有两个综合要素：一个充分运作的多用途试验生产设施和进行研究和工艺开发的能力。在过去的一年里，该单位进行了大约200种不同的大规模制备，包括：1）培养各种微生物，如重组大肠杆菌、重组酿酒酵母、重组巴斯德毕赤酵母、白喉棒状杆菌突变株、几种沙门氏菌和志贺氏菌以及炭疽芽孢杆菌突变株; 2）大量繁殖各种真核细胞如HeLa、CHO、果蝇和Sf-9; 3）纯化克量的白喉毒素CRM（交叉反应物质）和制备缀合疫苗所需的重组铜绿假单胞菌外毒素A。在研究和开发竞技场方面，该部门继续开展以下主题的工作：1）开发更有效的大规模蛋白质回收方法，在这里，我们进行了实验，实施了在离子交换剂，螯合和疏水树脂上捕获蛋白质的扩展床吸附技术; 2）进行旨在了解各种重组E.大肠杆菌菌株，试图找出为什么某些菌株更适合于使用微阵列技术和蛋白质分析生产外源蛋白质; 3）开发通过抑制蛋白水解活性从大肠杆菌表达敏感的重组梭菌蛋白质的发酵方法;和4）进行实验以改进从巴斯德毕赤酵母生产重组蛋白质，特别是内皮抑制素的方案。此外，该单位继续在两个主题的研究工作，一个是重组大肠杆菌的各种高密度生长策略对重组蛋白生产的影响，另一个是从HeLa/牛痘系统生产重组蛋白的过程。该单位与LIFE、NICHD合作，开展了开发新的伤寒沙门氏菌和福氏志贺菌疫苗的工作。这些产品目前正在进行实地试验。