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: 8515464
• 负责人: ROBERT B GENNIS
• 金额: $ 26.89万
• 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 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8515464
• 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 Targeting; 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; 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; screening; 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）：该项目的目的是为积分膜蛋白的生产提供一种新手段，这是通过X射线晶体学确定结构确定的良好候选者。结构信息对于了解酶和蛋白质的功能至关重要。不幸的是，那些嵌入膜中的蛋白质以纯形式和结晶更难获得。尽管膜蛋白可能占细胞中蛋白总数的30％，但膜蛋白的结构占蛋白质数据库中沉积的1％。膜蛋白对人类健康问题尤为重要，因为通过这些蛋白质，信息和物质会传播到各种细胞膜。尽管事实证明，哺乳动物起源的膜蛋白很难以结构研究所需的数量生产，但来自其他物种（包括微生物）的同源蛋白质更适合过度生产和纯化。长期以来，这是基础研究的成功策略。 我们的项目将专注于从嗜热微生物中获得适合70oC寿命的膜蛋白。这些生物的蛋白质在室温下往往非常稳定，与在较低温度下存在的生物的蛋白质相比，形成高质量晶体的可能性增加。在过去的几年中，其他群体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，我们的目标是适应这些极端热层的这些遗传工具，以过度生产其自己的膜蛋白，并附有亲和力标签。该方法将极大地促进样品的纯化，以实现结晶工作。 我们选择了三种不同的极端嗜热剂，它们将用于“生产”膜蛋白。我们将至少选择特别感兴趣的50种不同的蛋白质，并在嗜热中以亲和力标记的形式产生它们。这些蛋白质将被筛选为结晶条件，而那些最有前途的蛋白质将提供给膜蛋白结构中心，以进行完整结构确定的目标。 目前仅列出了大约218种膜蛋白结构。我们希望我们能展示大大增加这一数字的道路。 公共卫生相关性：绝大多数药物靶标是驻留在细胞或细胞内细胞内的膜中的蛋白质。不幸的是，这些蛋白质是最难获得的，从而限制了对结构数据的追求。这些人类蛋白中的许多蛋白质中的微生物中都有对应物在非常高温下适应生命的微生物，并且这些对应物特别稳定，易于获得和结晶。我们提出了一种获取这些热稳定蛋白的新方法，以确定其结构，从而推进与健康相关的生物医学研究。