SBIR Phase I: A Novel System for the Expression of Integral Membrane Proteins Using Halobacterium Halobium

SBIR 第一阶段：利用盐杆菌表达完整膜蛋白的新系统

• 批准号: 9660815
• 负责人: John Halladay
• 金额: $ 7.49万
• 依托单位: HaloGenetics, Inc
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 1997-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9660815&HistoricalAwards=false
• 关键词: SBIR; Phase; Novel; System; Expression

*** 9660815 Halladay This Small Business Innovation Research Phase I project will evaluate a novel method for the production of integral membrane proteins using the halophilic archeon Halobacterium halobium. Two key requirements for biochemical analysis of proteins involved in complex cellular processes are their production in quantity and their amenability to simple and rapid isolation. To circumvent limitations frequently encountered in native systems where insufficient amounts of protein are produced, recombinant DNA technology has been developed and used to produce soluble proteins in a variety of heterologous expression systems. Generally, however, these systems are not very efficient in expressing heterologous membrane proteins. Thus, a reliable heterologous production system for this class of proteins is needed. Halogenics, Inc. will construct the appropriate plasmid vectors and evaluate the usefulness of an H. halobium based membrane protein expression system. Several attributes of H. halobium indicate it will be an ideal system for production of membrane proteins. First, tools necessary for the routine introduction, maintenance, and expression of foreign DNA have already been developed for this organism. These include both a reliable transformation system for the introduction of DNA molecules, as well as plasmid vectors which can easily be modified and utilized as expression vectors. Second, H. halobium naturally produces very large amounts of bacteriorhodopsin, an integral membrane protein that is very similar in structure to the G-protein dependent receptor family--a major class of membrane proteins. H. halobium has a third advantage. It is readily , lysed by gentle, gradual dilution with distilled water or buffers to release the expressed proteins. In contrast, other recombinant systems inherently require denaturing conditions such as sonication, manual disruption, or the use of detergents to release expressed proteins. The proposed technology, combined with the low c osts associated with growing H. halobium and its easy lysis, will allow the inexpensive and simple production of integral membrane proteins in quantity. Such capability is of intense interest to researchers both in academic and applied disciplines. The purified proteins resulting from the use of this system would be used in drug discovery and development processes, diagnostic test kits, and therapeutic applications. ***

*** 9660815哈勒姆 这个小企业创新研究第一阶段项目将评估一种新的方法，使用嗜盐的archeon Halobacterium halobium生产完整的膜蛋白。 参与复杂细胞过程的蛋白质的生化分析的两个关键要求是它们的大量产生和它们对简单快速分离的顺从性。 为了避免天然系统中经常遇到的蛋白质产生量不足的限制，已经开发了重组DNA技术，并用于在各种异源表达系统中产生可溶性蛋白质。 然而，通常，这些系统在表达异源膜蛋白方面不是非常有效。 因此，需要用于这类蛋白质的可靠的异源生产系统。Halogenics，Inc.将构建合适的质粒载体并评估H.基于盐藻的膜蛋白表达系统。 H. Halobium表明它将是生产膜蛋白的理想系统。 首先，已经为这种生物体开发了常规引入、维持和表达外源DNA所需的工具。 这些包括用于引入DNA分子的可靠转化系统，以及可以容易地被修饰并用作表达载体的质粒载体。 第二，H.盐藻天然地产生非常大量的细菌视紫红质，这是一种在结构上与G蛋白依赖性受体家族（一种主要类型的膜蛋白）非常相似的完整膜蛋白。 H.盐生菌还有第三个优势。 通过用蒸馏水或缓冲液温和、逐渐稀释，其易于裂解以释放所表达的蛋白质。相比之下，其他重组系统本质上需要变性条件，例如超声处理、手动破碎或使用洗涤剂来释放表达的蛋白质。 提出的技术，结合低成本与增长H。盐菌及其易于裂解，将允许大量廉价和简单地生产完整的膜蛋白。 这种能力是在学术和应用学科的研究人员的强烈兴趣。 使用该系统得到的纯化蛋白质将用于药物发现和开发过程、诊断测试试剂盒和治疗应用。 ***