Preparation of biologically active platelet-derived growth factor isoforms AA and AB. Preferential formation of AB heterodimers.

摘要: We have expressed the mature platelet-derived growth factor (PDGF) A chain within a fusion protein of cro repressor and j-galactosidase in Escherichia coli. Monomeric PDGF-A was excised from this by CNBr cleavage. After protection thiols S-sulfonation, fragment purified gel permeation chromatography reversed-phase high-performance liquid chromatography. The monomeric dimerized presence mixture reduced oxidized glutathione to yield biologically active recombinant AA dimer (rPDGF-AA) with an overall about 0.2 mg/l culture. When rPDGF-A rPDGFB were reacted at stoichiometric concentrations glutathione, almost exclusively hetero-dimers type AB formed. Heterodimers stimulated [3H]thymidine incorporation into AKR-2B fibroblasts half-maximally 2 ng/ml. homodimers fivefold less active. About 60000 binding sites found for rPDGF-AB, 30000 rPDGF-AA 45 000 rPDGF-BB on fibroblasts. Platelet-derived is major mitogen serum which promotes proliferation smooth muscle cells vitro [I -31. In vivo, PDGF stored a-granules platelets it released following platelet activation. Purified cationic M, 30 exhibits considerable size heterogeneity species between 27 - 3 1 are thought result different processing, aging existence isoforms (type AA, AB, BB). biological activity all these forms destroyed chemical cleavage disulfide bridges. Mainly, heterodimers been human Amino acid sequencing data combination cDNA revealed that B chains partially identical. Interestingly, highly similar portion predicted amino sequence p28'-"'", transforming product simian sarcoma virus (SSV) [5