Purification and biochemical characterization of a soluble mouse interferon‐γ receptor produced in insect cells

• Published in: European journal of biochemistry Vol. 198; no. 2; pp. 441 - 450
• Main Authors: FOUNTOULAKIS, Michael, SCHLAEGER, Ernst‐Juergen, GENTZ, Reiner, JURANVILLE, Jean‐Francois, MANNEBERG, Michael, OZMEN, Laurence, GAROTTA, Gianni
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.06.1991; Blackwell
• Subjects: Amino Acids - analysis; Analysis of the immune response. Humoral and cellular immunity; Animals; Baculoviridae - genetics; Biological and medical sciences; Cell Division; Cell Line; Chromatography, Affinity; Chromatography, Gel; Drug Stability; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Glycosylation; Immunobiology; Immunoblotting; Interferon-gamma - metabolism; Kinetics; Mice; Microbiology; Miscellaneous; Molecular Weight; Moths; Receptors, Immunologic - genetics; Receptors, Immunologic - isolation & purification; Receptors, Immunologic - metabolism; Receptors, Interferon; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Regulatory factors and their cellular receptors; Transfection; Trypsin; Ligand binding; Heterologous system; Rodentia; Cytokine; Glycoproteins; Method; Baculovirus; Gene expression; Extracellular; Virus; Vertebrata; Membrane receptor; Mammalia; Baculoviridae; Mouse; Gamma interferon
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1991.tb16034.x

The extracellular domain of the mouse interferon γ receptor comprising amino acids 17–243 of the protein was produced in Spodoptera frugiperda cells infected with a recombinant baculovirus. The receptor was mainly secreted into the culture medium and was purified to homogeneity in several hundred milligram amounts. The purification procedure involved four chromatography steps and delivered a soluble and active receptor with an overall recovery of 30%. From each purification run, two pools of soluble receptor with the same interferon γ binding capacity were isolated. Under reducing electrophoretic conditions the protein of pool I migrates as two bands of molecular masses 32 and 34 kDa and of pool II as two bands of 30 and 32 kDa. The soluble receptor of both pools carries a heterogeneous glycosylation. After deglycosylation it appears as one protein band of 27 kDa. N‐linked carbohydrates contribute about 6 kDa and O‐linked carbohydrates 1 kDa to its molecular mass. The nonreduced protein specifically binds interferon γ on ligand blots and in a solid‐phase binding system and competes for the binding of radiolabeled interferon γ to the cell surface receptor. The soluble mouse interferon γ receptor exists as a monomer in physiological buffer and binds interferon γ in its dimeric form. It is stable at room temperature and against tryptic digestion, but is very sensitive to proteinase K digestion. The soluble mouse interferon γ receptor produced in the insect/baculovirus expression system may prove useful to study the function of interferon γ receptor as an antagonist of endogenous interferon γ in the treatment of immunological and inflammatory disorders.