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Production of soluble pMHC-I molecules in mammalian cells using the molecular chaperone TAPBPR.


ABSTRACT: 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.

SUBMITTER: O'Rourke SM 

PROVIDER: S-EPMC7451022 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Production of soluble pMHC-I molecules in mammalian cells using the molecular chaperone TAPBPR.

O'Rourke Sara M SM   Morozov Giora I GI   Roberts Jacob T JT   Barb Adam W AW   Sgourakis Nikolaos G NG  

Protein engineering, design & selection : PEDS 20191201 12


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 la  ...[more]

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