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 β‐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 60000 binding sites were found for rPDGF‐AB, 30000 for rPDGF‐AA and 45000 for rPDGF‐BB on AKR‐2B fibroblasts. Copyright © 1990, Wiley Blackwell. All rights reserved