Isolation and purification of a biologically active human platelet-derived growth factor BB expressed in Escherichia coli.
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 (hPDGF-B) was cloned into a plasmid under transcriptional control of the inducible E. coli Tac promoter. Expression of hPDGF-B from the final construct, pTacBIq, is regulated by the lactose repressor (LacIq). 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 form 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.
Alexander
D M
DM
Department of Biotechnology/Molecular Biology, Schering-Plough Research, Bloomfield, New Jersey 07003.
Hesson
T
T
Mannarino
A
A
Cable
M
M
Dalie
B L
BL
D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004926:Escherichia coli; D005347:Fibroblasts / Q000187:drug effects; D005822:Genetic Vectors; D006801:Humans; D008938:Mitosis / Q000187:drug effects; D008969:Molecular Sequence Data; D010982:Platelet-Derived Growth Factor / Q000096:biosynthesis / Q000302:isolation & purification* / Q000494:pharmacology; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification*
D010982:Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins; D004247:DNA; D017479:Receptors, Platelet-Derived Growth Factor