Preparation of biologically active platelet-derived growth factor isoforms AA and AB. Preferential formation of AB heterodimers.
We have expressed the mature platelet-derived growth factor (PDGF) A chain within a fusion protein of the cro repressor and beta-galactosidase in Escherichia coli. Monomeric PDGF-A was excised from this fusion protein by CNBr cleavage. After protection of thiols by S-sulfonation, this fragment was purified by gel permeation chromatography and reversed-phase high-performance liquid chromatography. The monomeric protein was dimerized in the presence of a mixture of reduced and oxidized glutathione to yield biologically active recombinant AA dimer (rPDGF-AA) with an overall yield of about 0.2 mg/l culture. When monomeric rPDGF-A and rPDGF-B were reacted at stoichiometric concentrations in the presence of glutathione, almost exclusively hetero-dimers of type AB were formed. Heterodimers AB stimulated [3H]thymidine incorporation into AKR-2B fibroblasts half-maximally at about 2 ng/ml. AA homodimers were fivefold less active. About 60,000 binding sites were found for rPDGF-AB, 30,000 for rPDGF-AA and 45,000 for rPDGF-BB on AKR-2B fibroblasts.
Hoppe
J
J
Institute for Physiological Chemistry, University of Würzburg, Federal Republic of Germany.
Weich
H A
HA
Eichner
W
W
Tatje
D
D
D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D003090:Coliphages / Q000235:genetics; D003488:Cyanogen Bromide; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000302:isolation & purification; D010982:Platelet-Derived Growth Factor / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D015183:Restriction Mapping
D046911:Macromolecular Substances; D010446:Peptide Fragments; D010982:Platelet-Derived Growth Factor; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D017479:Receptors, Platelet-Derived Growth Factor; D003488:Cyanogen Bromide