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.

描述（由申请人提供）：该项目的目的是提供一种新的方法来生产完整的膜蛋白，这些蛋白是通过X射线晶体学进行结构测定的良好候选物。结构信息对于了解酶和蛋白质的功能至关重要。不幸的是，那些包埋在膜中的蛋白质更难以获得纯的形式和结晶。虽然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，膜蛋白的结构占不到1%的沉积物。膜蛋白对人类健康问题特别重要，因为正是通过这些蛋白质，信息和物质才能穿过各种细胞膜。虽然哺乳动物来源的膜蛋白已被证明难以产生结构研究所需的数量，但来自其他物种（包括微生物）的同源蛋白更易于过量生产和纯化。长期以来，这一直是基础研究的成功策略。 我们的项目将专注于从适应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，并且与来自存在于较低温度下的生物体的蛋白质相比，形成优质晶体的可能性增加。在过去的几年里，其他研究小组已经学会了操纵这些生物的遗传学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，我们的目标是使这些遗传工具适应这些极端的超嗜热菌，以过量生产它们自己的带有亲和标签的膜蛋白。该方法将极大地促进样品的纯化，以使结晶的努力。 我们已经选择了三种不同的极端嗜热菌，我们将使用它们来“制造”膜蛋白。我们将挑选出至少50种不同的蛋白质，这些蛋白质是我们特别感兴趣的，并在嗜热菌中以亲和标记的形式生产它们。这些蛋白质将被筛选用于结晶条件，并且那些看起来最有希望的蛋白质将被提供给膜蛋白质结构中心以进行后续工作，以实现完整结构测定的目标。 目前仅列出了大约218种膜蛋白结构。我们希望我们能够展示一条大幅增加这一数字的道路。 公共卫生关系：绝大多数药物靶点都是位于细胞膜或细胞器周围的蛋白质。不幸的是，这些蛋白质是最难获得的，限制了对结构数据的追求。这些人类蛋白质中的许多在微生物中具有对应物，这些微生物适应于在非常高的温度下生活，并且这些对应物特别稳定，更容易获得和结晶。我们提出了一种新的方法来获得这些热稳定的蛋白质，以确定它们的结构，从而推进与健康相关的生物医学研究。