HUMAN TRANSCRIPTION FACTOR IMMUNOGENS: GENERATION OF A COMPLETE SET

人类转录因子免疫原：全套的生成

• 批准号: 8904907
• 负责人: Stephen Anderson
• 金额: $ 103.15万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-23 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8904907
• 关键词: Address; Affinity; Affinity Chromatography; Agreement; Antibodies; Antigens; Archives; Arizona; Atlases; Attention; Baculoviruses; Binding; Binding Proteins; Biological; Cells; Characteristics; Chromatin; Collection; Complex; Cytolysis; DNA; Databases; Deposition; Disease; Documentation; Electronics; Embryo; Ensure; Epitopes; Escherichia coli; Exhibits; Future; Generations; Genes; Genetic; Germ Cells; Goals; Human; Human Biology; Human Genome Project; Informatics; Insecta; Instruction; Laboratories; Malignant Neoplasms; Membrane; Metals; Mining; Monoclonal Antibodies; Occupations; Plasmids; Principal Investigator; Procedures; Process; Production; Proteins; Proteome; Protocols documentation; Reagent; Recombinant Proteins; Recombinants; Records; Residual state; Resources; Running; Sampling; Schedule; Scientist; Sequence Analysis; Shipping; Ships; Software Tools; Structure; System; Technology; Testing; Universities; Wheat; Work; Yeasts; base; chromatin immunoprecipitation; cost effective; design; design and construction; experience; expression vector; kidney cell; meetings; milligram; polypeptide; protein expression; repository; scale up; screening; success; three dimensional structure; transcription factor; vector; wasting

The goal of the proposed project is to assemble a collection of optimized immunogens based on the complete set of human transcription factors. These immunogens will be used by others to create renewable affinity reagents such as monoclonal antibodies. An initial, high priority appiication envisioned for such reagents is chromatin immunoprecipitation studies, and our aim is to design our immunogens so as to optimize them for this purpose. We will do this primarily using two strategies: (a) we will express transcription factor domains that flank the DNA recognition domain but do not overlap it in order to minimize inducing antibodies that might interfere with chromatin binding; and (b) wherever possible we will focus on transcription factor flanking domains that fold into complex three dimensional structures (native folds) and thus present highly specific 3D epitopes. We already have a protein production pipeline up and running that is producing > 1000 purified protein samples (including hundreds of human proteins) per year at the multi- milligram scale, so we are highly experienced in the relevant technologies, and meeting the goals of this FOA will require adding only a modest amount of extra capacity. We also have specialized software tools for the sophisticated analysis of human transcription factor sequences and the design of appropriate expression constructs. Our experience has shown that paying attention to the proper curation of genes and construct design at the beginning pays dividends downstream in terms of timely and cost-effective protein production because it minimizes wasting expensive wet lab resources on sub-par targets. Finally, we have a range of adaptable expression technologies that can be tailored to other classes of human protein targets (including secreted and membrane-bound proteins) in the future, so our approach is highly amenable to expressing additional sectors of the human proteome beyond transcription factors. RELEVANCE (See instmctions): The human genome project gave us a complete blueprint for the genetic instructions that control human biology. This proposal concerns a special set of human proteins known as "transcription factors", which are the master effector.proteins in the cell that have the job of interpreting these instructions and telling the cell what to do under all sets of conditions. In diseases such as cancer these instructions can become garbled, which is why it is important to understand how transcription factors work. This proposal concerns the creation of a specific collection of human transcription factor-derived proteins that will allow scientists to precisely interrogate which transcription factors address which sets of instructions under a variety of conditions, thus establishing the outlines of the master control circuits in human cells.

该项目的目标是组装一系列优化的免疫原 完整的人类转录因子集。这些免疫原将被其他人用来创造可再生的 亲和试剂，例如单克隆抗体。为此类设想的初始、高优先级应用程序 试剂是染色质免疫沉淀研究，我们的目标是设计我们的免疫原，以便 为此目的优化它们。我们将主要使用两种策略来做到这一点：（a）我们将表达 转录因子结构域位于 DNA 识别结构域两侧，但不与其重叠，以尽量减少 诱导可能干扰染色质结合的抗体； (b) 只要有可能，我们将重点关注 转录因子侧翼结构域折叠成复杂的三维结构（天然折叠）和 因此呈现出高度特异性的3D表位。我们已经建立并运行了一条蛋白质生产管道 每年在多工厂生产超过 1000 个纯化蛋白质样品（包括数百种人类蛋白质） 毫克级，因此我们在相关技术方面拥有丰富的经验，并且满足了这个目标 FOA 只需要添加少量的额外容量。我们还拥有专门的软件工具 人类转录因子序列的复杂分析和适当表达的设计 构造。我们的经验表明，关注基因的适当管理和构建 一开始的设计就可以及时且经济高效地生产蛋白质，从而为下游带来红利 因为它最大限度地减少了在低于标准目标上浪费昂贵的湿实验室资源。最后，我们有一系列 适应性表达技术，可以针对其他类别的人类蛋白质靶标（包括 分泌蛋白和膜结合蛋白）在未来，所以我们的方法非常适合表达 除转录因子之外的人类蛋白质组的其他部分。 相关性（参见说明）： 人类基因组计划为我们提供了控制人类的遗传指令的完整蓝图 生物学。该提案涉及一组特殊的人类蛋白质，称为“转录因子”， 是细胞中的主效应蛋白，负责解释这些指令并告诉 细胞在各种条件下要做什么。在癌症等疾病中，这些说明可以成为 乱码，这就是为什么了解转录因子如何工作很重要。该提案涉及 创建特定的人类转录因子衍生蛋白质集合，使科学家能够 精确询问哪些转录因子在各种条件下处理哪些指令集 条件，从而建立了人体细胞中主控制电路的轮廓。