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Preparation of recombinantly expressed T-cell Receptor-Protein α (TCRα) for structural studies

Thompson, Moriah
Ogunleye, Tiwalola
Cook, Gabriel A.

T-Cell Receptor Protein (TCR), a single-pass transmembrane protein found in T-cells, is involved in the signaling pathway in immune response. TCR binds antigens, activating the T-cell through signal transduction. The two chains of the receptor, alpha and beta, connect through a disulfide bond. While this is an essential protein for the immune system, the function and structure of TCR is not fully understood because the hydrophobic transmembrane region makes it difficult to study in typical sample conditions. Hydrophobic proteins like this receptor must be incorporated into detergent or lipid environments to run experiments that are commonly used to determine their function and structure. Our lab is developing methods to incorporate this hydrophobic protein into samples so that we can use Electron Resonance Spectroscopy (EPR) to study these properties. For this work, we are concentrating on expressing and purifying the alpha chain (TCRα). To run EPR on TCRα, we must attach a nitroxide group to the protein. TCRα was mutated at four positions, in four separate sequences, to replace a wild-type amino acid with a Cysteine residue. These Cysteines will form a thiol linkage with the nitroxide group. This presentation will explain the methods used to express, purify and concentrate Alanine 18 to Cysteine (A18C) and Lysine 14 to Cysteine (L14C), for the preparation of samples that can be measured by EPR.