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
基本信息
- 批准号:8318168
- 负责人:
- 金额:$ 27.72万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:ATP phosphohydrolaseATP-Binding Cassette TransportersAffinityArchaeaBacteriaBasic ScienceBiomassBiomedical ResearchCarrier ProteinsCellsCellular MembraneCellular StructuresCollectionCrystallizationCulture MediaDataDatabasesDepositionDetergentsDevelopmentDrug Delivery SystemsEnzymesEscherichia coliGeneticGenomeGoalsGrantGrowthHealthHigh temperature of physical objectHomologous ProteinHumanIntegral Membrane ProteinLearningLifeMembraneMembrane ProteinsMethodsMultienzyme ComplexesOrganellesOrganismPhaseProbabilityProductionProtein BiochemistryProteinsRecombinant ProteinsRecombinantsRelative (related person)ReportingSamplingScreening procedureSelenomethionineSolutionsSourceStructureSulfolobusSulfolobus acidocaldariusSulfolobus solfataricusSystemTemperatureThermusThermus thermophilusX-Ray Crystallographycold temperatureexpression vectorextreme thermophilefollow-upgene cloninghyperthermophileinterestmicroorganismnovelprotein functionprotein structurepublic health relevancerespiratory enzymestructural genomicssuccessthermophilic organismtool
项目摘要
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生产完整膜蛋白的新方法,该膜蛋白是通过 X 射线晶体学确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至关重要。不幸的是,那些嵌入膜中的蛋白质更难以以纯形式获得并结晶。尽管膜蛋白可能占细胞中蛋白质总数的 30%,但膜蛋白的结构仅占蛋白质数据库中沉积物的不到 1%。膜蛋白对人类健康问题特别重要,因为信息和物质正是通过这些蛋白质穿过各种细胞膜。虽然哺乳动物来源的膜蛋白已被证明难以生产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成功策略。 我们的项目将重点从适应 70oC 以上生活的嗜热微生物中获取膜蛋白。来自这些生物体的蛋白质在室温下往往相当稳定,与来自较低温度下存在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体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,我们的目标是为这些极端嗜热菌改造这些遗传工具,以过量生产带有亲和标签的自己的膜蛋白。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菌,用于“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形式生产它们。这些蛋白质将进行结晶条件筛选,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跟踪,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。我们希望能够展示一条显着增加这一数字的途径。
公共卫生相关性:绝大多数药物靶标是存在于细胞周围膜或细胞内细胞器中的蛋白质。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适应极高温度下生活的微生物中都有对应物,并且这些对应物特别稳定,更容易获得和结晶。我们提出了一种获得这些热稳定蛋白质的新方法,以确定其结构,从而推进与健康相关的生物医学研究。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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ROBERT B GENNIS其他文献
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Using extreme thermophiles for the homologous expression of membrane proteins
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