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

*** 9660815 Halladay这个小企业创新研究一期项目将评估一种利用嗜盐嗜盐菌生产整体膜蛋白的新方法。对参与复杂细胞过程的蛋白质进行生化分析的两个关键要求是它们的大量生产和它们易于简单快速地分离。为了避免在天然系统中经常遇到的产生蛋白质量不足的限制，重组DNA技术已经被开发出来，并用于在各种异种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效率不高。因此，需要一种可靠的这类蛋白的异源生产系统。Halogenics公司将构建合适的质粒载体，并评估基于盐芽孢杆菌的膜蛋白表达系统的有效性。盐藻的一些特性表明它将是生产膜蛋白的理想系统。首先，对于这种生物来说，常规导入、维持和表达外源DNA所必需的工具已经被开发出来。这包括一个可靠的转化系统来引入DNA分子，以及质粒载体，可以很容易地修饰和利用作为表达载体。其次，嗜盐菌自然产生大量的细菌视紫红质，这是一种完整的膜蛋白，在结构上与g蛋白依赖受体家族非常相似，g蛋白依赖受体是膜蛋白的主要类别。嗜盐菌还有第三个优势。用蒸馏水或缓冲液慢慢稀释，很容易裂解，释放出表达的蛋白质。相比之下，其他重组系统本质上需要变性条件，如超声、人工破坏或使用洗涤剂来释放表达的蛋白质。所提出的技术，结合低成本与生长的盐生双歧杆菌和它的容易裂解，将允许廉价和简单的生产整体膜蛋白的数量。这种能力引起了学术和应用领域研究人员的强烈兴趣。使用该系统获得的纯化蛋白将用于药物发现和开发过程、诊断测试试剂盒和治疗应用。* * *

*** 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. ***