Isolation of a Biologically Active Soluble Human Interferon-α Receptor-GST Fusion Protein Expressed in Escherichia coli

Abstract

The cDNA encoding the extracellular domain of the human interferon-α (IFN-α) receptor (Uzé, G., Lutfalla, G., and Gresser, I. Cell 1990;60:225–234) lacking the signal peptide has been expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The fusion protein represented 12% of total bacterial proteins and was found exclusively within cytoplasmic inclusion bodies. Inclusion body material was completely solubilized by 8 M urea; 20% solubilization was achieved by cell lysis in the presence of 0.45% cholamidopropyl dimethylammoniol-propane sulfonate and 1% Triton X-100. The soluble fusion protein was purified by gel filtration and affinity chromatography. Overall recovery of affinity purified fusion protein was approximately 100–200 μg/liter of cell culture. The affinity purified and refolded fusion protein exhibited the expected amino terminal sequence and M_r of 68,000 on reduced sodium dodecylsulfate gel electrophoresis. The protein reacted with antibodies specific for the cloned IFN-α receptor and inhibited the antiviral and antiproliferative activities of recombinant IFN-αB. We have demonstrated that the fusion protein binds to IFN-αB and competes with the cell surface receptor for binding to this IFN-α species.