HUMAN TRANSCRIPTION FACTOR IMMUNOGENS: GENERATION OF A COMPLETE SET

该项目的目标是根据免疫学原理组装一系列优化的免疫原。 一套完整的人类转录因子。这些免疫原将被其他人用来创造可再生的 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， 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，所述转录因子结构域位于DNA识别结构域的侧翼，但不与其重叠，以便最小化 诱导可能干扰染色质结合的抗体;和（B）只要可能，我们将关注 折叠成复杂三维结构（天然折叠）的转录因子侧翼结构域， 因此呈现高度特异性的3D表位。我们已经有了一个蛋白质生产管道， 每年生产> 1000个纯化蛋白质样品（包括数百个人类蛋白质）， 毫克规模，所以我们在相关技术方面经验丰富，并满足这一目标， FOA只需要增加少量的额外容量。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 结构。我们的经验表明，注意基因和结构的适当管理， 在及时和成本效益的蛋白质生产方面， 因为它最小化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，可针对其他类别的人类蛋白质靶（包括 分泌和膜结合蛋白），因此我们的方法非常适合表达 人类蛋白质组中转录因子以外的其他部分。 相关性（见说明）： 人类基因组计划给了我们一个完整的蓝图，控制人类的基因指令， 生物学这一提议涉及一组特殊的人类蛋白质，称为“转录因子”， 是细胞中的主要效应蛋白，它们负责解释这些指令， 细胞在各种条件下该做什么。在癌症等疾病中，这些指令可以成为 这就是为什么理解转录因子如何工作很重要。这项建议涉及 创建一个特定的人类转录因子衍生蛋白质集合，使科学家能够 精确地询问哪些转录因子在各种不同的条件下处理哪些指令集。 条件，从而建立人体细胞中的主控制电路的轮廓。

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.