Protein Production

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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.