Isolation and purification of a biologically active human platelet-derived growth factor BB expressed in Escherichia coli

Abstract Human platelet-derived growth factor (PDGF) was expressed in Escherichia coli from a high-level cytoplasmic expression vector. A cDNA fragment encoding the mature form of the human PDGF B chain (hPDGFB) was cloned into a plasmid under transcriptional control of the inducible E. coli Tac promoter. Expression of hPDGF-B from the final construct, pTacBI q , is regulated by the lactose repressor (LacI q ). Upon induction, a polypeptide of approximately 14 kDa that had the same molecular mass and immunoreactivity as authentic hPDGF-B was produced. The production of recombinant hPDGF-B was significantly increased in an E. coli strain (CAG629) defective in expression of the lon protease. Expression of hPDGF-B in the CAG629 strain accounted for approximately 1% of total cell protein. In this system, hPDGF-B is expressed as an insoluble, intracellular protein and can readily be obtained in a partially purified purm after differential centrifugation. Amino acid sequence determination of the purified protein has verified that the amino-terminal portion of the recombinant PDGF is correct. After renaturation into dimers, the purified recombinant hPDGF is fully functional in assays for receptor binding and mitogenesis.