PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF A SOLUBLE MOUSE INTERFERON-GAMMA RECEPTOR PRODUCED IN INSECT CELLS

The extracellular domain of the mouse interferon-gamma 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-gamma 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-gamma on ligand blots and in a solid-phase binding system and competes for the binding of radiolabeled interferon-gamma to the cell surface receptor. The soluble mouse interferon-gamma receptor exists as a monomer in physiological buffer and binds interferon-gamma 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-gamma receptor produced in the insect/baculovirus expression system may prove useful to study the function of interferon-gamma receptor as an antagonist of endogenous interferon-gamma in the treatment of immunological and inflammatory disorders.