HUMAN TRANSCRIPTION FACTOR IMMUNOGENS: GENERATION OF A COMPLETE SET

人类转录因子免疫原：全套的生成

基本信息

批准号：
8146979
负责人：
Stephen Anderson
金额：
$ 98.64万
依托单位：
RUTGERS, THE STATE UNIV OF N.J.
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：
2010-09-23 至 2013-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8146979
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 Screening procedure 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 success three dimensional structure transcription factor vector wasting

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 Screening procedure 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 success three dimensional structure transcription factor vector wasting

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项目摘要

DESCRIPTION (provided by applicant): 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 application 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: 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.

描述（由申请人提供）：拟议项目的目的是根据完整的人类转录因子集合一组优化的免疫原子。这些免疫原子将被其他人使用来创建可再生的亲和力试剂，例如单克隆抗体。染色质免疫沉淀研究是针对此类试剂的初始优先级应用，我们的目的是设计我们的免疫原子，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）我们将表达侧翼DNA识别域但不重叠的转录因子域，以最大程度地减少可能干扰染色质结合的抗体；（b）在可能的情况下，我们将重点放在折叠成复杂的三维结构（天然折叠）的转录因子侧面侧面域，从而呈现高度特定的3D表位。我们已经有一个蛋白质生产管道启动和运行，该管道每年在多毫克量表上每年生产> 1000个纯化的蛋白质样品（包括数百种人类蛋白质），因此我们在相关技术方面经验丰富，并且实现该FOA的目标将需要仅增加一定数量的额外容量。我们还拥有专门的软件工具，用于对人类转录因子序列进行复杂分析和适当表达构建体的设计。我们的经验表明，关注基因的适当策划和构造设计在一开始就在及时且具有成本效益的蛋白质生产方面向下游付费，因为它可以最大程度地减少浪费昂贵的湿实验室资源对低于标准的目标。最后，我们有一系列适应性的表达技术，这些技术可以在未来针对其他类别的人类蛋白质靶标（包括分泌和膜结合的蛋白质）量身定制，因此我们的方法高度适合表达人类蛋白质组的其他领域，超出转录因子。相关性：人类基因组项目为我们的控制人类生物学的遗传指示提供了完整的蓝图。该提案涉及一组被称为“转录因子”的特殊人蛋白，它们是细胞中的主效应蛋白，具有解释这些指示并告诉细胞在所有条件下该怎么做的工作。在诸如癌症之类的疾病中，这些指示可能会乱七八糟，这就是为什么了解转录因子的工作原理很重要的原因。该提案涉及创建特定人类转录因子衍生蛋白的特定集合，这些蛋白质将使科学家能够精确询问哪些转录因子解决了哪些条件下的指令集，从而确立了人类细胞中主控制电路的轮廓。