Production of platelet-derived growth factor receptor (PDGFR-beta) in E. coli. Mapping ligand binding domain.
Portions of the extracellular domain of the platelet-derived growth factor receptor beta (PDGFR-beta) were expressed as fusion proteins with a hexa His tag in E. coli. Following purification by Ni chelate chromatography, the recombinant receptors were tested in cross-competition studies with 125I-labelled PDGF-AA and -BB. Although of lower affinity than the native receptor (IC50 values of 10(-8) M) the recombinant molecules retained ligand binding specificity and neutralized the mitogenic effect of PDGF-BB. These data indicate that the ligand binding region lies within the first four immunoglobulin-like domains on PDGFR-beta. This E. coli expression system could be further used as a rapid and economical means to produce mutated receptors and map the ligand binding domain.
Rooney
B C
BC
Pharma Division, Preclinical Research, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Hosang
M
M
Hunziker
W
W
D001665:Binding Sites; D001667:Binding, Competitive; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006639:Histidine; D008970:Molecular Weight; D010449:Peptide Mapping; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D010982:Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins; D006639:Histidine; D017479:Receptors, Platelet-Derived Growth Factor