Protein Production

摘要 T细胞受体(TCR)赋予哺乳动物检测宿主细胞扰动的能力 由各种感染性病原体、物理损伤(热、辐射等)造成或癌前病变或 恶性细胞转化，同时避免可能诱发自身免疫的强烈自我反应。这个 最近的发现揭示了TCR及其发育前体的机械感受器特性， 前TCR在T细胞生物学中具有深远的意义。该计划项目的总体目标是 通过单分子、结构的聚合来理解TCR功能的分子基础 和生物调查。首先，核心B将通过提供大规模的蛋白质生产来支持每个项目 适用于单分子、单细胞、核磁共振或X射线结晶学。蛋白质的范围将是 以野生型、偶联或突变形式产生的包括：TcRαβ、TcRβ、前TcR(PTα/β)、MHCI(全长 和截短)，以及类似MHCI的T22、CD1c和CD1d。核心B协同运行，其中实验 (项目1-3)和计算团队(核心C)将与蛋白质生产团队合作指导 突变体设计、构建选择和后续生产。我们的团队已经为 此工作流程的成功演示。第二，核心B将作为DNA的中央存储库 帮助在项目之间转移知识的序列和构造。我们设计了一个矢量 便于每个克隆型TCR序列以及MHC序列在每个克隆之间转移的套件 计划项目中包含的技术。第三，核心B将与项目3密切合作，将 在原核生物和真核生物蛋白质生产过程中使用特定的标记技术。通过组合 这些关键功能，核心B将成为整个计划的中心枢纽。

ABSTRACT The T cell receptor (TCR) endows mammals with the capacity to detect cellular perturbations in a host resulting from myriad infectious pathogens, physical damage (thermal, irradiation, etc.) or pre-malignant or malignant cellular transformations while averting strong self-reactivities that could induce autoimmunity. The recent discoveries uncovering the mechanoreceptor properties of the TCR and its developmental precursor, the preTCR, have far-reaching implications in T cell biology. The overall goal of this Program Project is to understand the molecular underpinnings of TCR function through a convergence of single molecule, structural and biological investigations. First, Core B will support each project by providing protein production at scales appropriate for single molecule, single cell, NMR, or X-ray crystallography. The range of proteins to be produced in wild-type, conjugated, or mutated forms include: TCRαβ, TCRβ, preTCR (pTα/β), MHCI (full length and truncated), and MHCI-like T22, CD1c and CD1d. Core B operates synergistically, where the experimental (Project 1-3) and computational teams (Core C) will cooperate with the protein production team to guide mutant design, construct selection and subsequent production. Our teams have already laid the foundation for successful demonstration of this workflow. Second, Core B will serve as a central repository for DNA sequences and constructs to assist in transfer of knowledge between projects. We have designed a vector suite facilitating the transfer of each clonotypic TCR sequence as well as sequences of MHC between each technique encompassed in the Program Project. Third, Core B will work closely with Project 3 to incorporate specific labeling technologies into both prokaryotic and eukaryotic protein production processes. By combining these critical functions, Core B will be a central hub contributing to the entire program.