Production of soluble pMHC-I molecules in mammalian cells using the molecular chaperone TAPBPR.,Protein Engineering, Design and Selection

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Production of soluble pMHC-I molecules in mammalian cells using the molecular chaperone TAPBPR.
Protein Engineering, Design and Selection ( IF 2.6 ) Pub Date : 2020-07-29 , DOI: 10.1093/protein/gzaa015
Sara M O'Rourke ₁ , Giora I Morozov ₁ , Jacob T Roberts ₂ , Adam W Barb _{2,

3} , Nikolaos G Sgourakis ₁

Affiliation

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.

更新日期：2020-07-29

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