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.
Fountoulakis
M
M
Hoffmann-La Roche Ltd, Central Research Unit, Basel, Switzerland.
Schlaeger
E J
EJ
Gentz
R
R
Juranville
J F
JF
Manneberg
M
M
Ozmen
L
L
Garotta
G
G
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002455:Cell Division; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D015151:Immunoblotting; D007371:Interferon-gamma / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D009036:Moths; D011971:Receptors, Immunologic / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017471:Receptors, Interferon; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D014357:Trypsin
D000596:Amino Acids; D011971:Receptors, Immunologic; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D007371:Interferon-gamma; D014357:Trypsin