Expression of complete human IFN-gamma receptor and its extracellular domain in insect cells: purification and characterization of the recombinant proteins.
We here describe an efficient procedure for overexpression and purification of recombinant complete human interferon-gamma (IFN-gamma) receptor (IFN-gamma-R) and its extracellular fragment employing a baculovirus (BV) expression system. Infection of Sf 158 cells with recombinant BV results in membrane expression of high affinity IFN-gamma-R (Kd 1.6 x 10(-10) M), with approximately 10(6) molecules/cell 40 h postinfection. Solubilized, affinity-purified IFN-gamma-R and a secreted extracellular domain of IFN-gamma-R were compared for ligand-binding capacity and antagonistic activity in an IFN-gamma bioassay. Our results show that the complete receptor has a 2.5-fold higher ligand affinity and a 15-fold higher IFN-gamma in vitro-neutralizing capacity in an in vitro virus protection assay as compared to the extracellular fragment. This suggests that the transmembrane and cytoplasmic domains of IFN-gamma-R contribute to stability and/or enhance formation of biologically active receptor complexes in solution.
Moosmayer
D
D
Institute of Cell Biology and Immunology, University of Stuttgart, Germany.
Gerlach
E
E
Böttinger
H
H
Scheurich
P
P
Pfizenmaier
K
K
D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D002846:Chromatography, Affinity; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000378:metabolism; D008969:Molecular Sequence Data; D009036:Moths; D009500:Neutralization Tests; D017471:Receptors, Interferon / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D000096743:Interferon gamma Receptor
D017931:DNA Primers; D007371:Interferon-gamma; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D000096743:Interferon gamma Receptor