Figure 2. Production and characterization of soluble Mamu-DR αβ monomer and Mamu-DR*W201/P65 tetramer.

(a) Schematic presentation (cartoon) of the covalent peptide approach for developing class II MHC/peptide complex constructs. The epitope-coding sequence is linked to the 5′ Mamu-DRβ cDNA; Jun and Fos-BSP are introduced by linking the extracellular domains of DR-α and -β chain, respectively. The recombinant Mamu-DR αβ monomer is stabilized by leucine zipper (LZ) formed through Jun-Fos interaction. (b) Protein samples after 1^st round (lane 1) and second round (lane 2) purifications were separated in the SDS-PAGE gel in reduction conditions and stained. Lane 1, the (His)₆ tagged recombinant protein purified from Ni-NTA agarose; lane 2, the biotinylated Mamu-DR recombinants purified further through an avidin column after biotinylation. The arrow indicates two closely-separated protein bands in lane 2 (∼34, 36 kD) that correspond to predicted molecular weights of Mamu-DR α and β recombinants, respectively. (c) Dot blot assay indicates that anti-HLA-DR antibody (L243) bound to the soluble recombinant Mamu-DR αβ monomer purified by Ni-NTA affinity column (loading 1) and further by an avidin column (loading 2), but not to the denatured Mamu-DR αβ sample (loadings 3). The supernatant of non-transfected S2 cells served as a negative control (loading 4). (d) FPLC graph shows that the unbound Mamu-DR αβ molecules and free fluorescents were washed out, and the assembled Mamu-DR*W201-P65 tetramer was collected and marked as tetramer.