Novel affinity reagents for epigenetics markers
表观遗传学标记的新型亲和试剂
基本信息
- 批准号:7570180
- 负责人:
- 金额:$ 30.19万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2008
- 资助国家:美国
- 起止时间:2008-09-20 至 2010-07-31
- 项目状态:已结题
- 来源:
- 关键词:Abnormal CellAddressAffinityAnimalsAntibodiesAntigensArchitectureAreaBenchmarkingBindingBiological AssayCellsChimeric ProteinsClassCommunitiesComplexDissociationEngineeringEnhancersEpigenetic ProcessEpitopesEscherichia coliFigs - dietaryFoundationsGenerationsGoalsGoldGreen Fluorescent ProteinsGuidelinesHistonesImmunochemistryImmunoprecipitationIn VitroLeadLibrariesLysineMeasuresMethylationMonoclonal AntibodiesMutationNumbersPeptidesPerformancePlayPost-Translational Protein ProcessingProductionProtein EngineeringProteinsPublic HealthPurposeQuality ControlRangeReagentRecombinantsReproducibilityResearchSeriesSiteSpecificityStandards of Weights and MeasuresSurfaceTailTechniquesTechnologyTestingTimeTissuesTranscriptional ActivationUrinationVariantWestern Blottingbasecellular imagingchromatin immunoprecipitationcombinatorialconceptdesigndirected evolutionin vivoinnovationmonoclonal antibody productionnovelnovel strategiespolyclonal antibodyresearch studysuccess
项目摘要
DESCRIPTION (provided by applicant): Post-translational modifications (PTMs) of histone tails are major marks for transcription activation and silencing. Accordingly, detecting and capturing post-translationally modified histones represent a critical step in epigenetic research. A major technological bottleneck in this area of research is a paucity of high-quality affinity reagents. Polyclonal and monoclonal antibodies are the only widely available affinity reagents for histone tails, but they have fundamental limitations in reproducibility, scalability, storage and production throughput and expenses. The long-term goals of this project are to develop an innovative and powerful technology platform for facile production of high-quality affinity reagents for histone tails containing PTMs and to make a standard set of such affinity reagents broadly available to the epigenetics research community. This project is built on an innovative protein-engineering concept that our group has recently established. The concept, termed Affinity Clamping, harnesses the inherent specificity present in the so-called interaction domains and dramatically enhances their affinity and specificity by attaching an "enhancer domain" and subsequently optimizing its interaction interface by directed evolution of combinatorial libraries. The resulting affinity reagents with clamshell architecture, collectively termed "Affinity Clamps" thus "clamp" the target, leading to orders-of-magnitude higher affinity and specificity. Protein libraries made in this manner are predisposed to binding to a specific class of peptide motifs (e.g. histone tails with a methylated lysine), and they virtually guarantee successful engineering of high-performance affinity reagents for a predefined peptide motif. Affinity Clamping represents a paradigm shift in affinity reagent generation. Because Affinity Clamps are fully recombinant reagents produced in E. coli, they can be easily produced in large quantities and distributed. Also they can be reformatted into a variety of fusion proteins suitable for in vitro and in vivo applications. Our proof-of-concept experiments have successfully demonstrated the general feasibility of the Affinity Clamping concept and suggest its enormous potential. Because there exist a number of interaction domains that weakly bind to post-translationally modified histone tails, we are confident that we can apply the Affinity Clamping strategy to produce high-quality affinity reagents to a variety of histone motifs. The proposed project will critically evaluate the feasibility and potential of applying the Affinity Clamping technology to epigenetic histone marks. The specific aims of the initial project period are (i) to produce "Histone Clamps" for well-characterized histone lysine methylation sites and benchmark them against commercially available monoclonal antibodies for their performance in commonly used assays; and (ii) to produce Histone Clamps for histone methylation sites for which no high-quality antibodies exist. Such Histone Clamps will be provided to the epigenetic community, which will have a major impact on the quality, scale and types of epigenetics research.
PUBLIC HEALTH RELEVANCE: Accurately measuring the type and amounts of chemically modified forms of histones and capturing them for downstream analysis are major technological challenges in epigenetic research. This project will establish a totally new approach to facile generation of high-performance reagents for these purposes. This innovative and powerful technology will fill a major void in the currently epigenetic research, and products from this project, termed "Histone Clamps", will make it feasible to establish a standard set of epigenetic capture reagents that can be distributed broadly to the community and open new avenues of epigenetics research.
描述(由申请人提供):组蛋白尾部的翻译后修饰(PTM)是转录激活和沉默的主要标志。因此,检测和捕获后修饰的组蛋白代表了表观遗传学研究中的关键步骤。这一研究领域的一个主要技术瓶颈是缺乏高质量的亲和试剂。多克隆和单克隆抗体是唯一广泛可用的组蛋白尾部亲和试剂,但它们在再现性、可扩展性、储存和生产通量以及费用方面具有根本性限制。该项目的长期目标是开发一个创新的和强大的技术平台,用于轻松生产含PTM的组蛋白尾部的高质量亲和试剂,并为表观遗传学研究社区提供一套标准的亲和试剂。这个项目是建立在我们小组最近建立的创新蛋白质工程概念上的。该概念被称为亲和钳位(Affinity Clamping),利用了所谓的相互作用结构域中存在的固有特异性,并通过连接“增强子结构域”并随后通过组合文库的定向进化优化其相互作用界面来显著增强其亲和性和特异性。所得到的具有蛤壳结构的亲和试剂,统称为“亲和夹”,因此“夹”住靶标,导致更高数量级的亲和力和特异性。以这种方式制备的蛋白质文库倾向于结合特定类别的肽基序(例如具有甲基化赖氨酸的组蛋白尾部),并且它们实际上保证了针对预定肽基序的高性能亲和试剂的成功工程化。亲和夹持代表亲和试剂生成的范式转变。因为亲和夹是在大肠杆菌中生产的完全重组试剂。大肠杆菌,它们可以很容易地大量生产和分布。它们还可以被重新格式化为适合于体外和体内应用的各种融合蛋白。我们的概念验证实验已经成功地证明了亲和夹紧概念的一般可行性,并表明其巨大的潜力。由于存在许多与后修饰的组蛋白尾部弱结合的相互作用结构域,我们有信心我们可以应用亲和夹策略来生产针对各种组蛋白基序的高质量亲和试剂。拟议的项目将严格评估应用亲和钳技术的可行性和潜力表观遗传组蛋白标记。项目初期的具体目标是:(i)针对已充分表征的组蛋白赖氨酸甲基化位点生产“组蛋白夹”,并将其与市售单克隆抗体进行比较,以确定其在常用检测中的性能;以及(ii)针对没有高质量抗体的组蛋白甲基化位点生产组蛋白夹。这种组蛋白夹将提供给表观遗传学社区,这将对表观遗传学研究的质量,规模和类型产生重大影响。
公共卫生关系:准确测量化学修饰形式的组蛋白的类型和数量并捕获它们用于下游分析是表观遗传研究中的主要技术挑战。该项目将建立一种全新的方法来轻松生成用于这些目的的高性能试剂。这种创新和强大的技术将填补目前表观遗传学研究的主要空白,该项目的产品称为“组蛋白夹”,将使建立一套标准的表观遗传捕获试剂成为可能,这些试剂可以广泛分发给社区,并开辟表观遗传学研究的新途径。
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(1)
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SHOHEI KOIDE其他文献
SHOHEI KOIDE的其他文献
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