HUMAN TRANSCRIPTION FACTOR IMMUNOGENS: GENERATION OF A COMPLETE SET

描述（由申请人提供）：拟议项目的目标是基于完整的人类转录因子集合组装优化的免疫原集合。这些免疫原将被其他人用来产生可再生的亲和试剂，如单克隆抗体。一个初始的，高优先级的应用程序设想这样的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，以优化它们用于此目的。我们将主要使用两种策略来实现这一点：（a）我们将表达位于DNA识别结构域侧翼但不与其重叠的转录因子结构域，以使可能干扰染色质结合的诱导抗体最小化;以及（B）在可能的情况下，我们将关注转录因子侧翼结构域，其折叠成复杂的三维结构（天然折叠）并因此呈现高度特异性的3D表位。我们已经建立了一个蛋白质生产管道，每年以数毫克的规模生产> 1000个纯化蛋白质样品（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，满足本FOA的目标只需要增加少量的额外产能。我们还拥有专门的软件工具，用于人类转录因子序列的复杂分析和适当表达构建体的设计。我们的经验表明，在一开始就注意基因的正确管理和构建设计，在及时和具有成本效益的蛋白质生产方面为下游带来了好处，因为它最大限度地减少了在低于标准的目标上浪费昂贵的湿实验室资源。最后，我们有一系列可适应的表达技术，可以在未来针对其他类别的人类蛋白质靶点（包括分泌和膜结合蛋白）进行定制，因此我们的方法非常适合表达转录因子以外的人类蛋白质组的其他部分。 相关性：人类基因组计划为控制人类生物学的遗传指令提供了一个完整的蓝图。这项提议涉及一组特殊的人类蛋白质，称为“转录因子”，它们是细胞中的主要效应蛋白，负责解释这些指令并告诉细胞在所有条件下做什么。在癌症等疾病中，这些指令可能会变得混乱，这就是为什么了解转录因子如何工作很重要。该提案涉及创建一个特定的人类转录因子衍生蛋白质集合，这将使科学家能够精确地询问哪些转录因子在各种条件下处理哪些指令集，从而建立人类细胞中主控制电路的轮廓。

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.