Purification and crystallization of a complex between human interferon gamma receptor (extracellular domain) and human interferon gamma.
X-ray diffraction quality crystals have been obtained from a complex between interferon gamma and the extracellular domain of its high-affinity cell surface receptor. The crystals were obtained from interferon gamma/interferon gamma receptor complexes purified by size exclusion chromatography. Diffraction quality crystals required analyzing these complex samples by isoelectric focusing gels to select purified complex fractions devoid of unbound interferon gamma. These studies used interferon gamma receptor engineered with an eight amino acid N-terminal deletion to eliminate heterogeneity generated due to proteolytic cleavage. In addition, the receptor was expressed in an E. coli secretion cell line which eliminated the need to refold the protein. Hexagonal crystals were grown from 1.6 M ammonium phosphate solutions and belong to a spacegroup of P6(5)22 with unit cell dimensions a = 145.9 A and c = 180.3 A. These crystals diffract to at least 2.9 A resolution when exposed to synchrotron radiation. SDS PAGE analysis of the crystals demonstrated that both interferon gamma and the receptor were present. Analysis of the x-ray diffraction data revealed that the crystals contain complexes with a stoichiometry of 2:1 receptor: ligand within the crystallographic asymmetric unit and consist of approximately 55% solvent.
Windsor
W T
WT
Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.
Walter
L J
LJ
Syto
R
R
Fossetta
J
J
Cook
W J
WJ
Nagabhushan
T L
TL
Walter
M R
MR
D002850:Chromatography, Gel; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D017471:Receptors, Interferon / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D014961:X-Ray Diffraction
D017471:Receptors, Interferon; D007371:Interferon-gamma