Construction of soluble recombinant HLA-DR*1101-Ig and HLA-DR*1101-Bir molecules. Structure and homogeneity of purified soluble HLA-DR*1101 recombinant molecules. (a) Schematic representation of HLA-DR*1101-Ig and HLA-DR*1101-Bir constructs. The extracellular region of HLA-DR*1101β is fused with the Basic Zipper (BZ) and His tag. The HLA-DRα chain is fused with the Acid Zipper (AZ) and the human (h)IgG1 constant region or the biotynilation site BirA, respectively. All constructs are cloned into the pMT/V5/His Drosophila expression vector, in frame with the Drosophila leader sequence Bip, under the control of a metallotioneine promoter, inducible by CuSO4 addition. The HLA-DR*1101-Ig molecule is secreted into the supernatant of Drosophila cells as dimer of HLA-DR*1101α/β heterodimers, because of a disulphide bond between two hIgG domains. (b) SDS-PAGE of HLA-DR*1101-Ig molecules secreted into the supernatant of S2 cells (sup) after CuSO4 induction, and after purification by protein A affinity chromatography (Prot A). Gels were run either in reducing (+βm) or non-reducing (-βm) conditions. Indicated are the bands corresponding to the migration of the HLA-DRα-hIg homodimers, HLA-DRα-hIg monomers and HLA-DR*1101β-His tag proteins. (c) Western blotting analysis of HLA-DR*1101-Ig molecules, separated on SDS-PAGE as shown in (b). The blotted filter was probed at the same time with the anti-His tag and anti-hIg probes, as indicated in figure. Empty circle and asterisk indicate the migration of the HLA-DRα-hIg and HLA-DR*1101β-His tag, respectively. (d) SDS-PAGE analysis of the HLA-DR*1101-Bir molecule contained into the supernatant (sup) of S2 cells after CuSO4 induction, and after purification by immunoaffinity chromatography with L243 mAb (L243).