Production and isolation of recombinant somatomedin C

J Biochem. 1987 Jan;101(1):123-34. doi: 10.1093/oxfordjournals.jbchem.a121883.

Abstract

High-level production of a growth promoting peptide hormone somatomedin C (insulin-like growth factor I) has been achieved using recombinant DNA techniques in Escherichia coli. We found a new structural protein, designated as LH, to stabilize somatomedin C in vivo, and constructed expression vectors for somatomedin C fusing to LH through a methionine and through a tryptophan, respectively. Each of the fused proteins was produced at approximately 4.5 X 10(5) molecules per single E. coli cell and comprised more than 20% of the total cellular proteins. Somatomedin C was obtained in high yield by limited cyanogen bromide degradation of the methionine-type fused protein, in spite of somatomedin C itself having a Met at the 59th position, followed by renaturation of the resultant reduced peptide. The tryptophan-type fused protein was also converted to the peptide hormone by treating with 3-bromo-2-nitrophenylsulphenyl skatole or N-chlorosuccinimide. The recombinant somatomedin C obtained by these procedures was identical with the native one in amino acid sequence as well as in biological activity of stimulation of DNA synthesis in BALB/c 3T3 cells.

MeSH terms

  • Chromatography, High Pressure Liquid
  • Cyanogen Bromide
  • Escherichia coli / genetics
  • Genes, Synthetic
  • Indicators and Reagents
  • Insulin-Like Growth Factor I / biosynthesis*
  • Insulin-Like Growth Factor I / genetics
  • Insulin-Like Growth Factor I / isolation & purification
  • Luteinizing Hormone / genetics
  • Peptide Fragments / analysis
  • Plasmids
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Somatomedins / biosynthesis*

Substances

  • Indicators and Reagents
  • Peptide Fragments
  • Recombinant Proteins
  • Somatomedins
  • Insulin-Like Growth Factor I
  • Luteinizing Hormone
  • Cyanogen Bromide