{"doi":"10.1002/cpz1.36","title":"Production of Class II MHC Proteins in Lentiviral Vector‐Transduced HEK‐293T Cells for Tetramer Staining Reagents","abstract":"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.","journal":"Current Protocols","year":2021,"id":186310,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":22,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9557,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":744321,"name":"Vasanthi Ramachandiran","orcid":null,"position":1,"is_corresponding":false},{"id":744322,"name":"John Shires","orcid":null,"position":2,"is_corresponding":false},{"id":670467,"name":"Ge Bai","orcid":"0000-0001-7768-3119","position":3,"is_corresponding":false},{"id":744323,"name":"Kelly Jeter","orcid":null,"position":4,"is_corresponding":false},{"id":744324,"name":"Donielle L. Bell","orcid":null,"position":5,"is_corresponding":false},{"id":744325,"name":"Lixia Han","orcid":null,"position":6,"is_corresponding":false},{"id":744326,"name":"Tamara Kazarian","orcid":null,"position":7,"is_corresponding":false},{"id":744327,"name":"Kyla C. Ugwu","orcid":null,"position":8,"is_corresponding":false},{"id":489445,"name":"Oskar Laur","orcid":null,"position":9,"is_corresponding":false},{"id":744328,"name":"Susana Contreras‐Alcantara","orcid":null,"position":10,"is_corresponding":false},{"id":744329,"name":"Dale L. Long","orcid":null,"position":11,"is_corresponding":false},{"id":680026,"name":"John D. Altman","orcid":"0000-0001-9733-2359","position":12,"is_corresponding":false},{"id":680025,"name":"Richard A. Willis","orcid":"0000-0001-6890-1268","position":0,"is_corresponding":true}],"reference_count":68,"raw_metadata":null,"created_at":"2026-07-18T23:48:42.632147Z","pmid":"33539685","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}