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

Autor:	O'Rourke SM; Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, CA 95064, USA., Morozov GI; Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, CA 95064, USA., Roberts JT; Roy J Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA., Barb AW; Roy J Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA.; Department of Biochemistry and Molecular Biology, Complex Carbohydrate Research Center University of Georgia, Athens, GA 30602, USA., Sgourakis NG; Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, CA 95064, USA.
Jazyk:	angličtina
Zdroj:	Protein engineering, design & selection : PEDS [Protein Eng Des Sel] 2019 Dec 31; Vol. 32 (12), pp. 525-532.
DOI:	10.1093/protein/gzaa015
Abstrakt:	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. (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
Databáze:	MEDLINE
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