Production of Class II MHC Proteins in Lentiviral Vector-Transduced HEK-293T Cells for Tetramer Staining Reagents

Class II major histocompatibility complex peptide (MHC-IIp) multimers are precisely engineered reagents used to detect T cells specific for antigens from pathogens, tumors, and self-proteins. While the related Class I MHC/peptide (MHC-Ip) multimers are usually produced from subunits expressed in E....

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Bibliographic Details
Published in:	Current protocols Vol. 1; no. 2; p. e36
Main Authors:	Willis, Richard A, Ramachandiran, Vasanthi, Shires, John C, Bai, Ge, Jeter, Kelly, Bell, Donielle L, Han, Lixia, Kazarian, Tamara, Ugwu, Kyla C, Laur, Oskar, Contreras-Alcantara, Susana, Long, Dale L, Altman, John D
Format:	Journal Article
Language:	English
Published:	United States 01-02-2021
Subjects:	Escherichia coli HEK293 Cells Histocompatibility Antigens Class II Humans Indicators and Reagents Staining and Labeling protein engineering lentiviral transduction protein purification antigen-specific T cells Class II MHC tetramers flow cytometry protein expression Class II MHC multimers
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Summary:	Class II major histocompatibility complex peptide (MHC-IIp) multimers are precisely engineered reagents used to detect T cells specific for antigens from pathogens, tumors, and self-proteins. While the related Class I MHC/peptide (MHC-Ip) multimers are usually produced from subunits expressed in E. coli, most Class II MHC alleles cannot be produced in bacteria, and this has contributed to the perception that MHC-IIp reagents are harder to produce. Herein, we present a robust constitutive expression system for soluble biotinylated MHC-IIp proteins that uses stable lentiviral vector-transduced derivatives of HEK-293T cells. The expression design includes allele-specific peptide ligands tethered to the amino-terminus of the MHC-II β chain via a protease-cleavable linker. Following cleavage of the linker, HLA-DM is used to catalyze efficient peptide exchange, enabling high-throughput production of many distinct MHC-IIp complexes from a single production cell line. Peptide exchange is monitored using either of two label-free methods, native isoelectric focusing gel electrophoresis or matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry of eluted peptides. Together, these methods produce MHC-IIp complexes that are highly homogeneous and that form the basis for excellent MHC-IIp multimer reagents. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Lentivirus production and expression line creation Support Protocol 1: Six-well assay for estimation of production cell line yield Support Protocol 2: Universal ELISA for quantifying proteins with fused leucine zippers and His-tags Basic Protocol 2: Cultures for production of Class II MHC proteins Basic Protocol 3: Purification of Class II MHC proteins by anti-leucine zipper affinity chromatography Alternate Protocol 1: IMAC purification of His-tagged Class II MHC Support Protocol 3: Protein concentration measurements and adjustments Support Protocol 4: Polishing purification by anion-exchange chromatography Support Protocol 5: Estimating biotinylation percentage by streptavidin precipitation Basic Protocol 4: Peptide exchange Basic Protocol 5: Analysis of peptide exchange by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry Alternate Protocol 2: Native isoelectric focusing to validate MHC-II peptide loading Basic Protocol 6: Multimerization Basic Protocol 7: Staining cells with Class II MHC tetramers.
ISSN:	2691-1299
DOI:	10.1002/cpz1.36