Purification and biochemical characterization of a soluble human interferon gamma receptor expressed in Escherichia coli.
We purified and characterized a soluble human interferon gamma receptor expressed in Escherichia coli. The soluble receptor comprises the amino acids 15-246 of the encoded protein (Aguet, M., Dembic, Z., and Merlin, G. (1988) Cell 55, 273-280) and was purified from large scale fermentations through four chromatographic steps with an overall recovery of 28%. The refolded soluble receptor shows some heterogeneity on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, where it appears as the major band of 27 kDa molecular mass, accompanied by a few minor bands with molecular masses between 26 and 30 kDa. On reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis it appears as a homogeneous protein of 32 kDa molecular mass. The soluble interferon gamma receptor is an active and stable protein and is recognized by specific antibodies raised against the native receptor. When nonreduced it has the capacity to specifically bind interferon gamma and to compete for the binding of interferon gamma to the cell surface receptor. The observed heterogeneity of the soluble interferon gamma receptor under nonreducing electrophoretic conditions is probably due to different conformational forms resulting from the formation of non-native intramolecular disulfide bonds among the 8 cysteine residues present in the soluble interferon gamma receptor molecule.
Fountoulakis
M
M
Hoffmann-La Roche Ltd., Central Research Units, Basel, Switzerland.
Juranville
J F
JF
Stüber
D
D
Weibel
E K
EK
Garotta
G
G
D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting; D007371:Interferon-gamma / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D011971:Receptors, Immunologic / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017471:Receptors, Interferon; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D011971:Receptors, Immunologic; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D007371:Interferon-gamma