Characterisation of a secreted form of recombinant derived human growth hormone, expressed in Escherichia coli cells

J Pharm Biomed Anal. 1989;7(2):173-83. doi: 10.1016/0731-7085(89)80081-0.

Abstract

Recombinant DNA derived human growth hormone (rhGH), Genotropin, has been expressed in E. coli cells as a pre-hormone, where the heat stable enterotoxin II signal peptide (STII) was linked to hGH to get secretion of the hormone to the periplasmatic space. The pre-hormone was efficiently cleaved during secretion, by an endogenous signal peptidase generating the correct N-terminal (Phe) end as shown by protein sequence analysis. The purity of rhGH was studied by SDS-PAGE, in combination with laser densitometry and HI-HPLC. These techniques showed that the level of modified rhGH forms, e.g. aggregated and proteolytically cleaved (16 and 6 kDa) in the preparation was in the 0.5-1% range. Furthermore, evidence that the correct disulphide bonds (Cys53-Cys165; Cys182-Cys189) were formed in rhGH during secretion has been shown by a combination of tryptic fingerprint and amino acid analysis. CD-spectroscopic analysis suggested an identical secondary structure to that of pituitary derived human growth hormone (pit-hGH). Isoelectric focusing revealed an isoelectric point (pI) for rhGH of 5.0 similar to pit-hGH and in excellent agreement with the theoretical value 5.1, based on the primary sequence. Finally, an apparent molecular weight of 22,000 was obtained for rhGH, by SDS-PAGE. All these physico-chemical studies suggest that rhGH is structurally identical to pit-hGH, somatotropin.

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / chemistry
  • Chromatography, High Pressure Liquid
  • Circular Dichroism
  • Disulfides / analysis
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Gene Expression
  • Growth Hormone / analysis*
  • Growth Hormone / genetics
  • Growth Hormone / pharmacology
  • Humans
  • Isoelectric Focusing
  • Molecular Sequence Data
  • Molecular Weight
  • Protein Sorting Signals / chemistry
  • Protein Sorting Signals / genetics
  • Recombinant Proteins / analysis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / pharmacology

Substances

  • Amino Acids
  • Disulfides
  • Protein Sorting Signals
  • Recombinant Proteins
  • Growth Hormone