Interferon gamma receptor extracellular domain expressed as IgG fusion protein in Chinese hamster ovary cells. Purification, biochemical characterization, and stoichiometry of binding.
Agents that antagonize the functions of interferon gamma (IFN gamma) are potential pharmaceuticals against several immunological and inflammatory disorders. IFN gamma receptor-immunoglobulin G fusion proteins (IFN gamma R-IgG) function as antagonists of endogenous IFN gamma and have longer half-lives in vivo in comparison with soluble IFN gamma receptors (sIFN gamma R), consisting of the extracellular region of the native sequence. A fusion protein comprising the extracellular domain of the human IFN gamma receptor and the hinge, CH2 and CH3 domains of the human IgG3 constant region, was expressed in Chinese hamster ovary cells. The IFN gamma R-IgG3 fusion protein was secreted into the culture medium as a 175-kDa glycoprotein and was purified over Protein G-Sepharose, DEAE-Sepharose, and size exclusion chromatography. IFN gamma R-IgG3 bound IFN gamma in solid phase assays and ligand blots, competed for the binding of radiolabeled IFN gamma to the cell surface receptor of Raji cells, and inhibited the IFN gamma-mediated antiviral activity with an efficiency at least one order of magnitude higher than that of the soluble receptor produced in the same expression system. Two IFN gamma R-IgG3 fusion proteins bound two IFN gamma dimers forming a complex of approximately 380 kDa. In immunodiffusion assays, the IFN gamma R-IgG3 fusion protein did not precipitate IFN gamma. Dissociation of bound IFN gamma from IFN gamma R-IgG3 was 2-fold slower than from the sIFN gamma R produced in insect cells.
Fountoulakis
M
M
F. Hoffmann-La Roche Ltd., Department of Gene Technology, Basel, Switzerland.
Mesa
C
C
Schmid
G
G
Gentz
R
R
Manneberg
M
M
Zulauf
M
M
Dembic
Z
Z
Garotta
G
G
D000818:Animals; D016466:CHO Cells; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics* / Q000378:metabolism; D007371:Interferon-gamma / Q000378:metabolism; D011485:Protein Binding; D017471:Receptors, Interferon / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D000096743:Interferon gamma Receptor
D007074:Immunoglobulin G; D007371:Interferon-gamma; D017471:Receptors, Interferon; D011993:Recombinant Fusion Proteins; D000096743:Interferon gamma Receptor