Production of Class II MHC Proteins in Lentiviral Vector-Transduced HEK-293T Cells for Tetramer Staining Reagents
© 2021 Wiley Periodicals LLC..
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.
| Errataetall: | |
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| Medienart: |
E-Artikel |
| Erscheinungsjahr: |
2021 |
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| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:1 |
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| Enthalten in: |
Current protocols - 1(2021), 2 vom: 09. Feb., Seite e36 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Willis, Richard A [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 17.06.2021 Date Revised 11.02.2024 published: Print ErratumIn: Curr Protoc. 2022 Aug;2(8):e552. - PMID 36005902 Citation Status MEDLINE |
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| doi: |
10.1002/cpz1.36 |
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| Förderinstitution / Projekttitel: |
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| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Class II MHC multimers | |
| 650 | 4 | |a Class II MHC tetramers | |
| 650 | 4 | |a antigen-specific T cells | |
| 650 | 4 | |a flow cytometry | |
| 650 | 4 | |a lentiviral transduction | |
| 650 | 4 | |a protein engineering | |
| 650 | 4 | |a protein expression | |
| 650 | 4 | |a protein purification | |
| 650 | 7 | |a Histocompatibility Antigens Class II |2 NLM | |
| 650 | 7 | |a Indicators and Reagents |2 NLM | |
| 700 | 1 | |a Ramachandiran, Vasanthi |e verfasserin |4 aut | |
| 700 | 1 | |a Shires, John C |e verfasserin |4 aut | |
| 700 | 1 | |a Bai, Ge |e verfasserin |4 aut | |
| 700 | 1 | |a Jeter, Kelly |e verfasserin |4 aut | |
| 700 | 1 | |a Bell, Donielle L |e verfasserin |4 aut | |
| 700 | 1 | |a Han, Lixia |e verfasserin |4 aut | |
| 700 | 1 | |a Kazarian, Tamara |e verfasserin |4 aut | |
| 700 | 1 | |a Ugwu, Kyla C |e verfasserin |4 aut | |
| 700 | 1 | |a Laur, Oskar |e verfasserin |4 aut | |
| 700 | 1 | |a Contreras-Alcantara, Susana |e verfasserin |4 aut | |
| 700 | 1 | |a Long, Dale L |e verfasserin |4 aut | |
| 700 | 1 | |a Altman, John D |e verfasserin |4 aut | |
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