Open AccessProduction of soluble pMHC-I molecules in mammalian cells using the molecular chaperone TAPBPR
Author(s) -
Sara M. O’Rourke,
Giora I. Morozov,
Jacob T. Roberts,
Adam W. Barb,
Nikolaos G. Sgourakis
Publication year - 2019
Publication title -
protein engineering, design and selection
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.627
H-Index - 109
eISSN - 1741-0134
pISSN - 1741-0126
DOI - 10.1093/protein/gzaa015
Subject(s) - major histocompatibility complex , chaperone (clinical) , chinese hamster ovary cell , mhc restriction , antigen , biology , antigen processing , polyclonal antibodies , mhc class i , human leukocyte antigen , microbiology and biotechnology , t cell receptor , computational biology , peptide , chemistry , t cell , biochemistry , immune system , genetics , cell culture , medicine , pathology
Current approaches for generating major histocompatibility complex (MHC) Class-I proteins with desired bound peptides (pMHC-I) for research, diagnostic and therapeutic applications are limited by the inherent instability of empty MHC-I molecules. Using the properties of the chaperone TAP-binding protein related (TAPBPR), we have developed a robust method to produce soluble, peptide-receptive MHC-I molecules in Chinese Hamster Ovary cells at high yield, completely bypassing the requirement for laborious refolding from inclusion bodies expressed in E.coli. Purified MHC-I/TAPBPR complexes can be prepared for multiple human allotypes, and exhibit complex glycan modifications at the conserved Asn 86 residue. As a proof of concept, we demonstrate both HLA allele-specific peptide binding and MHC-restricted antigen recognition by T cells for two relevant tumor-associated antigens. Our system provides a facile, high-throughput approach for generating pMHC-I antigens to probe and expand TCR specificities present in polyclonal T cell repertoires.