Preparation of biologically active platelet-derived growth factor type BB from a fusion protein expressed in Escherichia coli.
Preparations of the mitogen platelet-derived growth factor (PDGF) from human platelets contain two related polypeptides termed A chain and B chain. PDGF-B is highly homologous to a portion of p28v-sis, the transforming protein of simian sarcoma virus. We have studied the mitogenic potential of a PDGF-BB-like homodimer by expressing the sequence coding for the mature part of PDGF-B in Escherichia coli. Expression was achieved as cro-beta-gal-PDGF-B fusion protein which was exclusively found in the "inclusion bodies". A monomeric PDGF-B fragment shortened by 12 amino acid residues from the NH2 terminus was excised from the 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. This monomeric protein was dimerized in the presence of a mixture of reduced and oxidized glutathione to yield biologically active rPDGF-BB with an overall yield of approximately 0.7 mg of rPDGF-BB/L of culture. Escherichia coli rPDGF-BB stimulated [3H]thymidine incorporation into AKR2B fibroblast at concentrations of about 1 ng/mL.
Hoppe
J
J
Department of Cytogenetics, GBF--Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, FRG.
Weich
H A
HA
Eichner
W
W
D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003488:Cyanogen Bromide; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010957:Plasmids; D010982:Platelet-Derived Growth Factor / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000494:pharmacology
D010446:Peptide Fragments; D010982:Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins; D003488:Cyanogen Bromide