Expression of biologically active human pre-procorticotropin releasing hormone in E. coli: characterization and purification

Int J Biochem. 1990;22(11):1341-9. doi: 10.1016/0020-711x(90)90318-w.

Abstract

1. Human pre-procorticotropin releasing hormone (CRH) was expressed in E. coli strain TG2 as a fusion protein with beta-galactosidase. 2. A 140 kDa band which corresponded to beta-galactosidase pre-proCRH fusion protein was identified in lysates of TG2 cells harbouring the recombinant plasmid pre-proCRH (10-196) [ph PPC (10-196)] after sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Coomassie Blue staining. The identity of the fusion protein was confirmed by Western blotting and a two-site immunoradiometric assay. 3. Purification of the fusion protein from isolated, washed and solubilized inclusion bodies was achieved by ion-exchange chromatography in the presence of 8 M urea. 4. When comparing the adrenocorticotropin-releasing activity on a molar basis, the potency of the chimeric CRH precursor was 4% of that of synthetic r/h CRH (1-41).

MeSH terms

  • Cloning, Molecular
  • Corticotropin-Releasing Hormone / biosynthesis
  • Corticotropin-Releasing Hormone / genetics*
  • Corticotropin-Releasing Hormone / isolation & purification
  • Escherichia coli / enzymology*
  • Humans
  • Immunoblotting
  • Immunoradiometric Assay
  • Protein Denaturation
  • Protein Precursors / biosynthesis
  • Protein Precursors / genetics*
  • Protein Precursors / isolation & purification
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Solubility
  • beta-Galactosidase / biosynthesis
  • beta-Galactosidase / genetics

Substances

  • Protein Precursors
  • Recombinant Fusion Proteins
  • pro-corticotropin releasing hormone
  • Corticotropin-Releasing Hormone
  • beta-Galactosidase