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Expression and Functional Characterization of Recombinant HLA-DM.

Miguel Álvaro-Benito1,2,3, Jakob Holzapfel4, Christian Freund4

  • 1Department of Immunology, Ophthalmology, ENT, School of Medicine, Universidad Complutense de Madrid, Madrid, Spain. migalv07@ucm.es.

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|January 1, 2026
PubMed
Summary
This summary is machine-generated.

This study details reproducible protocols for producing recombinant Human Leukocyte Antigen-DM (HLA-DM) and HLA-DR proteins. These methods enable functional analysis of HLA-DM

Keywords:
Antigen presentationFluorescence polarizationHLA-DMMHC IIPeptide editingPeptide exchange

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Area of Science:

  • Immunology
  • Molecular Biology
  • Protein Biochemistry

Background:

  • Human Leukocyte Antigen-DM (HLA-DM) is a nonclassical MHC class II molecule crucial for antigen presentation.
  • It catalyzes peptide exchange on MHC class II molecules, ensuring stable peptide-MHC complexes for CD4+ T cells.
  • Reliable production of recombinant HLA-DM and HLA-DR is essential for studying their function.

Purpose of the Study:

  • To provide detailed protocols for the production of recombinant HLA-DM and HLA-DR proteins.
  • To outline methods for the functional analysis of recombinant HLA-DM.
  • To establish a foundation for mechanistic studies on catalyzed peptide exchange.

Main Methods:

  • Utilized a baculovirus expression system in Spodoptera frugiperda (Sf9) cells for protein production.
  • Employed bacmid preparation, baculovirus amplification, and large-scale protein expression.
  • Purified proteins using affinity and size exclusion chromatography.
  • Assessed HLA-DM activity via fluorescence polarization-based peptide exchange assays.

Main Results:

  • Established step-by-step protocols for recombinant HLA-DM and HLA-DR production.
  • Achieved high-purity protein yields through optimized purification strategies.
  • Demonstrated functional evaluation of HLA-DM activity using peptide exchange assays.

Conclusions:

  • The described protocols enable reproducible production of recombinant HLA-DM and HLA-DR.
  • These methods provide a reliable resource for researchers investigating antigen presentation and T cell interactions.
  • Facilitates further functional and mechanistic studies on catalyzed peptide exchange by HLA-DM.