Protein Production

蛋白质生产

• 批准号: 10020598
• 负责人: Robert Joseph Mallis
• 金额: $ 40.11万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-29 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10020598
• 关键词: Amino Acids; Autoimmunity; Biological; Biology; Cell Lineage; Cells; Cellular biology; DNA; DNA Repository; DNA Sequence; Development; Foundations; Future; Goals; Human Resources; Individual; Investigation; Knowledge; Label; Length; Malignant - descriptor; Mammals; Mechanoreceptors; Molecular; Mutate; Peptide/MHC Complex; Process; Production; Property; Protein Engineering; Proteins; Resources; Stable Isotope Labeling; Structure; T-Cell Receptor; T-Lymphocyte; Techniques; Technology; Work; X-Ray Crystallography; design; design and construction; irradiation; metaplastic cell transformation; mutant; pathogen; premalignant; programs; receptor function; repository; single molecule; vector

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.

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 由无数传染性病原体、物理损伤（热、辐射等）或癌前病变， 恶性细胞转化，同时避免可能诱导自身免疫的强烈自身反应。的 最近的发现揭示了TCR及其发育前体的机械感受器特性， preTCR对T细胞生物学具有深远的影响。本计划项目的总体目标是 了解TCR功能的分子基础，通过单一分子，结构 和生物学调查。首先，核心B将通过提供规模化的蛋白质生产来支持每个项目 适用于单分子、单细胞、NMR或X射线晶体学。蛋白质的范围 以野生型、缀合或突变形式产生的TCR包括：TCRαβ、TCRβ、preTCR（pTα/β）、MHCI（全长 和截短的）和MHCI样T22、CD 1c和CD 1d。核心B协同操作，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， 突变体设计、构建体选择和随后的生产。我们的团队已经为 成功演示了此工作流程。第二，核心B将作为DNA的中央储存库 序列和结构，以协助项目之间的知识转移。我们设计了一个载体 一套促进每个克隆型TCR序列以及MHC序列在每个克隆型TCR序列之间转移的设备， 项目中包含的技术。第三，核心B将与项目3密切合作， 将特异性标记技术应用于原核和真核蛋白质生产过程。通过组合 核心B将成为整个计划的中心枢纽。