Using extreme thermophiles for the homologous expression of membrane proteins

使用极端嗜热菌进行膜蛋白的同源表达

• 批准号: 8150453
• 负责人: ROBERT B GENNIS
• 金额: $ 27.72万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8150453
• 关键词: ATP phosphohydrolase; ATP-Binding Cassette Transporters; Affinity; Archaea; Bacteria; Basic Science; Biomass; Biomedical Research; Carrier Proteins; Cells; Cellular Membrane; Cellular Structures; Collection; Crystallization; Culture Media; Data; Databases; Deposition; Detergents; Development; Drug Delivery Systems; Enzymes; Escherichia coli; Genetic; Genome; Goals; Grant; Growth; Health; High temperature of physical object; Homologous Protein; Human; Integral Membrane Protein; Learning; Life; Membrane; Membrane Proteins; Methods; Multienzyme Complexes; Organelles; Organism; Phase; Probability; Production; Protein Biochemistry; Proteins; Recombinant Proteins; Recombinants; Relative (related person); Reporting; Sampling; Screening procedure; Selenomethionine; Solutions; Source; Structure; Sulfolobus; Sulfolobus acidocaldarius; Sulfolobus solfataricus; System; Temperature; Thermus; Thermus thermophilus; X-Ray Crystallography; cold temperature; expression vector; extreme thermophile; follow-up; gene cloning; hyperthermophile; interest; microorganism; novel; protein function; protein structure; public health relevance; respiratory enzyme; structural genomics; success; thermophilic organism; tool

DESCRIPTION (provided by applicant): The purpose of the project is to provide a new means for the production of integral membrane proteins that are good candidates for structure determination by X-ray crystallography. Structural information is essential to understand how enzymes and proteins function. Unfortunately, those proteins that are embedded in membranes are much more difficult to obtain in pure form and to crystallize. Although membrane proteins may comprise as much as 30% of the total number of proteins in a cell, the structures of membrane proteins make up less than 1% of depositions in the Protein Data Bank. Membrane proteins are particularly important to human health issues, since it is through these proteins that information as well as substances pass across various cellular membranes. While membrane proteins of mammalian origins have proven to be difficult to produce at quantities necessary for structural studies, homologous proteins from other species, including microorganisms, are more amenable to overproduction and purification. This has long been a successful strategy for basic research. Our project will focus on obtaining membrane proteins from thermophilic microorganisms that are adapted to life above 70oC. Proteins from these organisms tend to be quite stable at room temperature, and the probability of forming good quality crystals is increased in comparison to proteins from organisms that exist at lower temperatures. In the past few years, other groups have learned to manipulate the genetics of these organisms for the production of soluble proteins. Utilizing our expertise in membrane protein biochemistry, we aim to adapt these genetic tools for these extreme hyperthermophiles to overproduce their own membrane proteins with affinity tags attached. This method will greatly facilitate the purification of sample to enable crystallization efforts. We have selected three different extreme thermophiles that we will use to "manufacture" membrane proteins. We will pick at least 50 different proteins that are of particular interest, and produce them in affinity-tagged form within the thermophile. These proteins will be screened for crystallization conditions, and those that appear the most promising will be provided to the Center for Structures of Membrane Proteins to follow-up with the goal of complete structure determination. There are only about 218 structures of membrane proteins currently listed. We hope we can demonstrate a path towards significantly increasing this number. PUBLIC HEALTH RELEVANCE: The large majority of drug targets are proteins that reside in the membrane that surrounds our cells or organelles within our cells. Unfortunately, these proteins are the most difficult to obtain, limiting the pursuit of structural data. Many of these human proteins have counterparts in microorganisms that are adapted to life at very high temperatures, and these counterparts are particularly stable, easier to obtain, and to crystallize. We propose a new way to obtain these thermally stable proteins for the purposes of determining their structures, thus advancing health-related biomedical research.

项目描述（由申请人提供）：该项目的目的是提供一种生产完整膜蛋白的新方法，该蛋白是x射线晶体学测定结构的良好候选者。结构信息对于理解酶和蛋白质的功能是必不可少的。不幸的是，那些嵌入在膜中的蛋白质很难以纯形式获得和结晶。尽管膜蛋白可能占细胞中蛋白质总数的30%，但在蛋白质数据库中，膜蛋白的结构只占沉积物的不到1%。膜蛋白对人类健康问题尤其重要，因为信息和物质正是通过这些蛋白质穿过各种细胞膜的。虽然哺乳动物起源的膜蛋白已被证明难以生产出结构研究所需的数量，但来自其他物种（包括微生物）的同源蛋白更容易过量生产和纯化。长期以来，这一直是基础研究的一个成功策略。我们的项目将侧重于从适应70℃以上生活的嗜热微生物中获得膜蛋白。来自这些生物体的蛋白质在室温下往往相当稳定，与存在于较低温度下的生物体的蛋白质相比，形成高质量晶体的可能性增加了。在过去的几年里，其他研究小组已经学会了操纵这些生物体的基因来生产可溶性蛋白质。利用我们在膜蛋白生物化学方面的专业知识，我们的目标是适应这些极端嗜热生物的遗传工具，以过量生产带有亲和力标签的膜蛋白。这种方法将大大方便样品的纯化，使结晶的努力。我们选择了三种不同的极端嗜热菌，我们将用它们来“制造”膜蛋白。我们将挑选至少50种特别感兴趣的不同蛋白质，并在嗜热菌中以亲和标记的形式生产它们。这些蛋白将被筛选为结晶条件，那些看起来最有希望的将提供给膜蛋白结构中心进行跟踪，以完成结构确定的目标。目前仅列出了218种膜蛋白结构。我们希望我们能够展示一条显著增加这一数字的道路。