Isolation of a biologically active soluble human interferon-alpha receptor-GST fusion protein expressed in Escherichia coli.
The cDNA encoding the extracellular domain of the human interferon-alpha (IFN-alpha) 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 micrograms/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-alpha receptor and inhibited the antiviral and antiproliferative activities of recombinant IFN-alpha B. We have demonstrated that the fusion protein binds to IFN-alpha B and competes with the cell surface receptor for binding to this IFN-alpha species.
Division of Cytokine Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.
N Y
NY
Nguyen
D
D
Sackett
R D
RD
Hirata
D E
DE
Levy
J C
JC
Enterline
J B
JB
Bekisz
M H
MH
Hirata
D000998:Antiviral Agents / Q000494:pharmacology; D001667:Binding, Competitive; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D007370:Interferon Type I / Q000494:pharmacology; D016898:Interferon-alpha; D017510:Protein Folding; D053633:Receptor, Interferon alpha-beta; D017471:Receptors, Interferon / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins; D012995:Solubility
D000998:Antiviral Agents; D007370:Interferon Type I; D016898:Interferon-alpha; D017471:Receptors, Interferon; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C076799:interferon-alpha 8; D053633:Receptor, Interferon alpha-beta; D005982:Glutathione Transferase