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

Protein Expr Purif. 1992 Jun;3(3):204-11. doi: 10.1016/1046-5928(92)90016-p.

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 (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.

MeSH terms

  • Base Sequence
  • DNA / genetics
  • Escherichia coli
  • Fibroblasts / drug effects
  • Genetic Vectors
  • Humans
  • Mitosis / drug effects
  • Molecular Sequence Data
  • Platelet-Derived Growth Factor / biosynthesis
  • Platelet-Derived Growth Factor / isolation & purification*
  • Platelet-Derived Growth Factor / pharmacology
  • Receptors, Platelet-Derived Growth Factor / metabolism
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / isolation & purification*

Substances

  • Platelet-Derived Growth Factor
  • Recombinant Fusion Proteins
  • DNA
  • Receptors, Platelet-Derived Growth Factor