Simple, rapid, high-purity preparation of recombinant human platelet-derived growth factor-BB

Biotechnol Lett. 2007 Sep;29(9):1333-9. doi: 10.1007/s10529-007-9411-9. Epub 2007 Jun 2.

Abstract

Recombinant human platelet-derived growth factor-BB (rhPDGF-BB) is used to treat full-thickness diabetic ulcers and is being investigated for use in other chronic ulcers, non-healing wounds, and periodontal defects. A simple, novel method for expression and purification of rhPDGF-BB from Escherichia coli is now described. This method produces the dimeric protein in high yield (10-12 mg/g wet cell mass) and with a purity >95%. rhPDGF-BB was exclusively found in inclusion bodies (IBs) representing approx. 30% of the total cell proteins. The IBs were extracted and the monomer purified by RP-HPLC. The purified rhPDGF-B monomer was then refolded using Tris buffer and subsequently dimerized to produce biologically active rhPDGF-BB. This product was composed of two polypeptide chains, each approx. 12 kDa. The final product exhibited specific activity in a fibroblast proliferation assay indistinguishable from that of the WHO reference standard.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Becaplermin
  • Cell Proliferation / drug effects
  • Cells, Cultured
  • Cloning, Molecular / methods
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Fibroblasts / cytology
  • Fibroblasts / drug effects*
  • Fibroblasts / physiology
  • Genetic Enhancement / methods
  • Humans
  • Platelet-Derived Growth Factor / administration & dosage
  • Platelet-Derived Growth Factor / chemistry
  • Platelet-Derived Growth Factor / genetics
  • Platelet-Derived Growth Factor / metabolism
  • Protein Engineering / methods*
  • Proto-Oncogene Proteins c-sis
  • Recombinant Proteins / metabolism

Substances

  • Platelet-Derived Growth Factor
  • Proto-Oncogene Proteins c-sis
  • Recombinant Proteins
  • Becaplermin