EXPRESSION AND CHARACTERIZATION OF RECOMBINANT HUMAN INTERFERON ALPHA RECEPTOR

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The long-term goal of this study, initiated in June, 1991, is to express and characterize the protein encoded by the recently isolated cDNA for the putative Type I interferon receptor. Currently, very little is known about the biochemical and functional nature of the receptor protein. The plasmid containing the cDNA has been obtained from American Type Culture Collection. The full-length insert, coding for the interferon receptor, has been converted, by site-directed mutagenesis, into a cDNA encoding a soluble form of this receptor by changing an amino acid condon into a stop condon just prior to the transmembrane region. This insert was subcloned into a eukaryotic expression vector called pdR (obtained from T. Kishimoto, Osaka University, Japan) for expression in dihydrofolate reductase -negative Chinese hamster ovary cells (DG44, from L. Chasin, Columbia University, NY). The entire cDNA has been sequenced to confirm the changes made. Efforts to generate a soluble form of the receptor protein are underway. The protein products will be purified and used to generate both polyclonal and monoclonal antibodies to the protein. In addition, experiments will be performed to assess the capacity of this protein to bind various species of human interferon-alpha readily available in our laboratory. These studies should lead to an enhanced understanding of ligand-receptor interactions in the interferon system. Corollary studies are also underway to attempt expression in bacterial systems (primarily in Dr. Nguyen's laboratory) and to make antibodies to these products.