Plant-Based Production of Human Major Histocompatibility Complex Class II Molecules.

Major histocompatibility complex class II (MHC II) protein triggers adaptive immune responses by presenting antigens on antigen-presenting cells (APCs) to be recognised by CD4+ T cells. Recombinant MHC II proteins are effective for the analysis of peptide-binding interaction, detection of antigen-specific T-cells and clinical testing for donor-specific anti-human leukocyte antigen (HLA) antibody for organ transplantation. However, Escherichia coli-based production is limited due to difficulty in obtaining properly folded protein complexes. In this study, we utilised the expression machinery of Nicotiana benthamiana via the Tsukuba System to produce the soluble extracellular domains of MHC II proteins. Thirteen different MHC II proteins of the three isotypes (HLA-DR, DQ and DP) conjugated with peptides (pMHC II) were successfully expressed. The HLA-DRA*01:01/DRB1*01:01 covalently linked to a class II-associated invariant chain peptide (CLIP) or influenza hemagglutinin (HA, 306-318) was further analysed as a model. The resultant proteins were detectable in soluble fractions and purified using metal affinity purification and ion exchange chromatography, yielding 4.37 and 2.81 μg/gFW, respectively. pMHC II were also biotinylated in vitro using commercial BirA, enabling future construction of tetramers or multimers. The purified proteins were confirmed through enzyme-linked immunosorbent assay (ELISA) using anti-HLA antibodies. Furthermore, Surface plasmon resonance biosensor (Biacore) verified glycosylation-dependent recognition of MHC II-HA complex by cognate T-cell receptor (HA1.7 TCR), resulting in an equilibrium dissociation constant of KD = 3.62 ± 1.59 μM. Overall, this plant-based system offers a promising scalable and economical alternative for large-scale production of functional MHC II proteins for basic and clinical applications.