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TITLE: A NEW METHOD FOR THE PRODUCTION OF MHC CLASS II/PEPTIDE COMPLEXES ASSOCIATED BY FOS AND JUN DIMERIZATION DOMAINS

Junbao Yang,¹ Ruili Shi,¹ Jeremy Goodman,¹ T. Mohanakumar.<sup>1

1</sup>Surgery, Washington University School of Medicine, St. Louis, MO, USA

The goal of this study was to create high–level expression of major histocompatibility complex (MHC) class II/peptide complexes for generation of MHC class II/peptide tetramers. We designed HLA–DR/peptide fusion proteins with Fos and Jun dimerization domains attached at the C–termini of HLA–DR &agr; and &bgr; chains to facilitate chain pairing. In addition, the binding peptides were covalently attached at the N–termini of HLA–DR &bgr; chains by a flexible linker, and a Bir A enzyme substrate peptide (BSP) was fused to the C–terminus of the HLA–DR &agr; chain for site–specific biotinylation and tetramerization with streptavidin. The fusion genes were constructed by primer dimer formation PCR and multiple–round overlapping PCR. Stable insect cell expression cell lines were established by co–transfection of Drosophila Melanogaster–2 cells with HLA–DR &agr; and &bgr; chain expression vectors and a blasticidin resistant gene expression vector and selection with blasticidin. The expressed proteins were purified by anti–HLA–DR affinity column and biotinylated with Bir A enzyme. Three HLA–DR/peptide complexes, HLA–DR2/myelin basic protein_(85–99), HLA–DR7/HLA–A29_(234–249) and HLA– DR7/HLA–B44_(62–77) were successfully expressed at 20 mg/L, 2 mg/L and 1.5 mg/L, respectively. The proteins were purified to more than 95% homogeneity by single round affinity column purification. Our results suggested that the production of MHC class II/peptide complexes may be limited by chain pairing of MHC &agr; and &bgr; chains. Attachment of the BSP tag on the C–terminus of &agr; chains had minimal influence on chain pairing. The HLA–DR/peptide complex fusion proteins could be biotinylated specifically and efficiently with Bir A enzyme for tetramerization by streptavidin. Binding specificity of the tetramer is currently being analyzed. We conclude that fusing Fos and Jun dimerization domains to &agr; and &bgr; chains, respectively, as well as attaching a BSP tag to the C–terminus of the &agr; chain, results in better production of MHC class II/peptide complexes for the purpose of generating MHC class II tetramers. This study may provide a general strategy for production of MHC class II tetramers for immunological applications.