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(申请人提供)：该项目的目的是为生产完整的膜蛋白提供一种新的方法，这些蛋白可以很好地用于X射线结晶学结构测定。结构信息对于了解酶和蛋白质的功能是必不可少的。不幸的是，那些镶嵌在膜中的蛋白质更难以纯形式获得和结晶。虽然膜蛋白可能占细胞中蛋白质总数的30%，但膜蛋白的结构在蛋白质数据库中的沉积中所占比例不到1%。膜蛋白对人类健康问题特别重要，因为正是通过这些蛋白，信息和物质才能通过各种细胞膜传递。虽然哺乳动物来源的膜蛋白被证明很难生产出结构研究所需的数量，但来自其他物种的同源蛋白，包括微生物，更容易过度生产和纯化。长期以来，这一直是基础研究的一项成功战略。我们的项目将专注于从适应70摄氏度以上生活的嗜热微生物中获得膜蛋白。这些生物体的蛋白质在室温下往往相当稳定，与在较低温度下存在的生物体的蛋白质相比，形成高质量晶体的可能性增加。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，我们的目标是改造这些基因工具，使这些极端嗜热菌能够过度生产自己的膜蛋白，并附上亲和力标签。这种方法将极大地促进样品的提纯，使结晶努力成为可能。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力标记的形式生产它们。将对这些蛋白质的结晶条件进行筛选，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进行后续研究，以实现完全结构确定的目标。目前，膜蛋白的结构只有218种左右。我们希望我们能够展示一条大幅增加这一数字的道路。 与公共健康相关：绝大多数药物靶标是存在于我们细胞或细胞内细胞器周围的膜中的蛋白质。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都有对应的蛋白质，这些蛋白质适应于在非常高的温度下生活，而且这些蛋白质特别稳定，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，以确定其结构，从而推进与健康相关的生物医学研究。