Studies were conducted to express and purify the extracellular domain and subdomains of human IFN-a receptor (HuIFN-R) and to generate antibodies for the investigation of signal transduction mechanisms. Expression and Purification of HuIFN-R: The gene encoding the extracellular domain of HuIFN-R was expressed as fusion with glutathione-S-transferase (GST). The GST-Receptor was isolated from inclusion bodies in the absence (soluble form) and presence (insoluble form) of 8 M urea. Overall recovery was approximately 1 mg/liter of cell culture. Both insoluble and soluble forms inhibited the antiviral and antiproliferative activities of 5 -10 U of IFN alpha and showed a Kd value of approximately 10-9 M in receptor-ligand binding assays. The binding of pyrene-labeled IFN-aB to the purified receptor proteins was measured using fluorescence polarization. Results indicated a saturable increase in the fluorescence anisotropy with increasing concentrations of receptor proteins. As expected, reduced binding was obtained by preincubating the labeled IFN- aB with a 10-fold excess of unlabeled IFN. Soluble Form of GST-Receptor: Induction of protein expression with 0.1 mM IPTG at 30oC, sequential disruption with french press in Tris pH 9.0 buffer containing DTT, EDTA, PMSF and CHAPS (or Triton) released HuIFN-R in soluble form from inclusion bodies. Approximately 20% of HuIFNR was obtained in the supernatant of cell lysate. Eighty per cent of the expressed HUIFNR still remained in the inclusion bodies and were solubilized only in the presence of 8M urea. Expression and Isolation of HuIFN-R Subdomains: Three subdomains, named M1, M2 and M3 (residues 1-103, 93-260 and 224- 401) were expressed in PRSET vector and served to identify monoclonal antibodies which reacted with specific epitopes found in each fragment. Of the three subdomains, only two, M2 and M3 appeared to exhibit antiviral and antiproloferative activities. Further, only M3 exhibited saturable binding to pyrene-labeled IFN-aB with a Kd comparable to the Kd which was determined for the purified receptor fusion-proteins. Antibody Production: Polyclonal antibodies against GST-receptor produced 25 - 30% inhiition of both IFN alpha binding to receptor and IFN biological activity. Several monoclonal antibodies immunoprecipitated the natural cellular receptor and specifically recognized the cellular receptor on western blots. All the projects have been completed. Findings from these investigations will significantly enrich our understanding of the mechanisms by which interferons and other cytokines modulate immune responses and control viral and neoplastic growth through their receptors.
