HUMAN TRANSCRIPTION FACTOR IMMUNOGENS: GENERATION OF A COMPLETE SET

拟议项目的目标是基于以下条件集合优化的免疫原 全套人类转录因子。这些免疫原将被其他人用来创造可再生的 亲和试剂，如单抗。一种初始的、高优先级的应用程序，用于 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，请对它们进行优化。我们将主要使用两种策略来做到这一点：(A)我们将表示 转录因子结构域在DNA识别结构域的两侧，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；及(B)在可能的情况下，我们将专注于 转录因子侧翼结构域折叠成复杂的三维结构(天然折叠)和 从而呈现高度特异的3D表位。我们已经有了一条蛋白质生产管道并在运行 每年生产1000个纯化蛋白质样本(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，并达到了这一目标 FOA将只需要增加适度的额外容量。我们还拥有专门的软件工具，用于 人类转录因子序列的精细分析和适当表达的设计 构造。我们的经验表明，注意适当的基因调控和构建 在一开始的设计在及时和经济高效的蛋白质生产方面为下游带来红利 因为它最大限度地减少了将昂贵的湿实验室资源浪费在低于标准的目标上。最后，我们有一系列 适应性表达技术，可针对其他类别的人类蛋白质靶标(包括 分泌型和膜结合型蛋白)，所以我们的方法是高度服从表达的 人类蛋白质组的转录因子以外的其他部分。 相关性(请参阅说明)： 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。这项建议涉及一组特殊的人类蛋白质，称为“转录因子”，它 是主效应器。细胞中的蛋白质负责解释这些指令并告诉 单元格在所有条件下都要做什么。在癌症等疾病中，这些指令可能会变成 这就是为什么了解转录因子是如何工作的重要原因。这项提议涉及到 创造一组特定的人类转录因子衍生蛋白，将使科学家能够 精确地询问哪些转录因子处理各种不同的指令集 条件，从而建立了人体细胞中主控制电路的轮廓。

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.