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HLA-DM is a nonclassical MHC class II molecule that plays an essential role in antigen presentation by catalyzing peptide exchange on MHC class II molecules, ensuring the presentation of stable peptide-MHC complexes to CD4+ T cells. This chapter describes detailed protocols for the production and functional analysis of recombinant HLA-DM and HLA-DR proteins. Using a baculovirus expression system in Spodoptera frugiperda (Sf9) cells, we outline step-by-step methods for bacmid preparation, baculovirus amplification, and large-scale protein expression. Comprehensive purification strategies, including affinity chromatography and size exclusion chromatography, are provided to ensure high-purity protein yields. Additionally, we describe functional assays, utilizing fluorescence polarization-based peptide exchange to evaluate HLA-DM activity. These protocols enable the reproducible production of recombinant HLA-DM and HLA-DR, providing a reliable foundation for researchers conducting functional and mechanistic studies on catalyzed peptide exchange.

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Expression and Functional Characterization of Recombinant HLA-DM

  • 实验方案

  • 发表日期：

    2026年

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  • Miguel Álvaro-Benito,
  • Jakob Holzapfel,
  • Christian Freund

摘要

HLA-DM is a nonclassical MHC class II molecule that plays an essential role in antigen presentation by catalyzing peptide exchange on MHC class II molecules, ensuring the presentation of stable peptide-MHC complexes to CD4+ T cells. This chapter describes detailed protocols for the production and functional analysis of recombinant HLA-DM and HLA-DR proteins. Using a baculovirus expression system in Spodoptera frugiperda (Sf9) cells, we outline step-by-step methods for bacmid preparation, baculovirus amplification, and large-scale protein expression. Comprehensive purification strategies, including affinity chromatography and size exclusion chromatography, are provided to ensure high-purity protein yields. Additionally, we describe functional assays, utilizing fluorescence polarization-based peptide exchange to evaluate HLA-DM activity. These protocols enable the reproducible production of recombinant HLA-DM and HLA-DR, providing a reliable foundation for researchers conducting functional and mechanistic studies on catalyzed peptide exchange.