Expression of a rat PDGF receptor beta ectodomain generates a low affinity ligand antagonist.
Platelet-derived growth factor (PDGF) controls migration and proliferation of mesenchymal cells and is thought to be involved in the pathophysiology of different diseases such as arteriosclerosis and tumorigenesis. In order to investigate the role of PDGF in rat models for such diseases, sufficient amounts of PDGF antagonists are needed. For this purpose we expressed the extracellular domain (ectodomain) of the rat PDGF receptor beta (PDGFR beta) in insect cells using a baculovirus vector. A hydrophilic octapeptide was added onto the N-terminus of the receptor ectodomain to follow its expression with specific anti-FLAG antibodies. The FLAG tag was also utilized to design a rapid two-step purification protocol. Purified FLAG-tagged rat PDGFR beta ectodomain had an affinity to PDGF-BB identical to the untagged ectodomain as determined by Scatchard analysis. FLAG-tagged PDGFR beta ectodomain in solution, however, did not compete for PDGF-BB binding to full length cellular receptors at concentrations expected for an high affinity antagonist.
Herren
B
B
Pharma Division, F. Hoffmann-La Roche Ltd., Basel.
Pech
M
M
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D004247:DNA / Q000235:genetics; D004195:Disease Models, Animal; D009036:Moths / Q000235:genetics; D016296:Mutagenesis / Q000235:genetics; D009842:Oligopeptides / Q000235:genetics; D051381:Rats; D017479:Receptors, Platelet-Derived Growth Factor / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D012995:Solubility
D009842:Oligopeptides; D011994:Recombinant Proteins; D004247:DNA; D017479:Receptors, Platelet-Derived Growth Factor