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High-Throughput Production of Class I MHC Tetramers for Immunological Studies Using Chaperone-Mediated Peptide
Hoang Anh T Phan1,2,3, Nikolaos G Sgourakis4,5,6
1Center for Computational and Genomic Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
Abstract:
The production of Major Histocompatibility Complex (MHC) Class I proteins bound to different peptides of interest is essential for many immunological studies. However, the preparation of MHC-I molecules, whether wild-type or engineered, requires in vitro refolding with a high-affinity peptide, which stabilizes the complex, leading to a sufficient protein yield. This presents a challenge for subsequent peptide exchange reactions, commonly used to generate pMHC-I tetramer libraries. Here, we describe an optimized protocol for chaperone-mediated exchange of a high-affinity placeholder peptide for an array of target peptides. Using this protocol, a pMHC-I tetramer library encompassing 135 different antigen specificities can be produced within 3 days.
