CORE--TECHNICAL CORE

核心--技术核心

• 批准号: 7537249
• 负责人: WOLFGANG H FISCHER
• 金额: $ 55.07万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-12-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7537249
• 关键词: ACVR1 gene; Activins; Affinity; Amino Acids; Animals; Antibodies; Antibody Formation; Antigens; Binding; Binding Proteins; Biological; Blocking Antibodies; Blood capillaries; C-terminal; Carrier Proteins; Cells; Chemicals; Chimeric Proteins; Chinese Hamster Ovary Cell; Circular Dichroism; Complementary DNA; Complex; Consult; Controlled Study; Coupling; Development; Electrophoresis; Endoglin; Equipment; Estradiol; Event; Extracellular Domain; Family member; Follicle Stimulating Hormone; Follistatin; Gel; Gene Proteins; Generations; Glutathione S-Transferase; Goals; Gonadal Steroid Hormones; Growth and Development function; High Pressure Liquid Chromatography; Human Resources; Immune Sera; Immunoassay; Immunoblotting; Immunohistochemistry; Immunoprecipitation; In Vitro; Individual; Lead; Ligands; Liquid substance; Mass Spectrum Analysis; Measurement; Membrane; Methods; Molecular Conformation; Mus; NMR Spectroscopy; Nature; Numbers; Pattern; Peptide Synthesis; Peptides; Phase; Physical condensation; Physiological; Pituitary Gland; Pituitary Hormones; Plasma; Post-Translational Protein Processing; Preparation; Principal Investigator; Procedures; Production; Progesterone; Proteins; Proteoglycan; Protocols documentation; Quality Control; Radioimmunoassay; Reagent; Recombinant Activin; Recombinant Proteins; Recombinants; Regulation; Research Personnel; Role; Sampling; Sepharose; Sequence Analysis; Services; Signal Transduction; Solid; Standardization; Steroids; Structure; Structure-Activity Relationship; System; TGF beta type III receptor; Techniques; Testosterone; Training; UMOD gene; Update; activin A; activin B; capillary; cell growth; cost; cross reactivity; design; disulfide bond; immunoaffinity chromatography; improved; inhibin; inhibin B; inhibin receptor; instrumentation; interest; method development; novel; polypeptide; programs; protein expression; receptor; receptor binding; reproductive function; research study; sex; synthetic peptide; tissue culture; urinary gonadotropin fragment

This Program Project focuses on the role of activins and inhibins and their membrane-bound receptors, soluble binding proteins and modulators in the regulation of reproductive function, cellular growth and development. The primary focus of this Program is to characterize the physical interactions and molecular events that lead to signal propagation by activin and inhibin. The second focus is to explore the physiologic and pathophysiologic significance of these molecules at the cellular and system levels and to study the control of protein expression and secretion as well as modes of action. The Technical Core (Core Component B) provides vital services, including protein expression, peptide synthesis, peptide/protein characterization, and generation of antibodies, needed by the Projects of this Program to carry out structural, functional and mechanistic studies. Large quantities of recombinant activin, inhibin and betaglycan fragments, essential for the structural and biological studies, will be expressed by the Core. Peptides for generation of antisera and for specific blockers of antibodies will be synthesized and purified by the Core. Synthetic peptides and recombinant proteins generated by the Core, additional recombinant proteins produced by individual Projects, and novel proteins isolated by this Program will be characterized. Posttranslational modifications of ligands and/or receptors will be identified. The analysis of polypeptides will be achieved using a number of techniques including HPLC, mass spectroscopy, NMR and automated Edman degradation. Synthetic peptides conjugated to carrier proteins will be used as immunogens to develop antibodies directed towards gene products predicted from cDNA sequences. Alternatively, proteins produced using in vitro cell expression systems will be injected directly into host animals for production of antisera. To reduce cross-reactivity with antigens other than the protein of interest, selected antisera will be immunoaffinity purified when necessary. Core B will additionally provide immunoassays of pituitary gonadotropic hormones and sex steroids for physiologic and pharmalogic studies proposed. The reagents and services provided require specialized equipment and highly trained personnel which can be most efficiently consolidated in a Core. In addition, the integrated nature of the Core assures cost efficiency, standardization of protocols and facilitates quality control.

本项目的重点是激活素和抑制素及其膜结合受体、可溶性结合蛋白和调节剂在生殖功能、细胞生长和发育调节中的作用。该计划的主要重点是描述通过激活素和抑制素导致信号传播的物理相互作用和分子事件。第二个重点是探索这些分子在细胞和系统水平上的生理学和病理生理学意义，并研究蛋白质表达和分泌的调控以及作用方式。技术核心(核心组件B)提供本计划项目所需的重要服务，包括蛋白质表达、多肽合成、多肽/蛋白质表征和抗体生成，以进行结构、功能和机制研究。大量重组激活素、抑制素和β-葡聚糖片段将由Core表达，这对结构和生物学研究是必不可少的。用于产生抗血清和特定抗体阻滞剂的多肽将由Core合成和提纯。核心产生的合成肽和重组蛋白，个别项目产生的额外重组蛋白，以及本计划分离的新蛋白将被表征。将确定配体和/或受体的翻译后修饰。多肽的分析将使用许多技术，包括高效液相色谱、质谱学、核磁共振和自动埃德曼降解。与载体蛋白结合的合成肽将被用作免疫原，以开发针对从cdna序列预测的基因产物的抗体。或者， 使用体外细胞表达系统产生的蛋白质将直接注射到宿主动物中，用于生产抗血清。为了减少与感兴趣蛋白质以外的抗原的交叉反应，选定的抗血清将在必要时进行免疫亲和纯化。CORE B还将为拟议中的生理学和药理学研究提供脑下垂体促性腺激素和性类固醇的免疫分析。所提供的试剂和服务需要专门的设备和训练有素的人员，这些可以最有效地整合到一个核心中。此外，核心的综合性质确保了成本效益、协议的标准化和促进质量控制。