Production of bioactive human hemangiopoietin in Escherichia coli

Biochemistry (Mosc). 2010 Apr;75(4):481-5. doi: 10.1134/s0006297910040127.

Abstract

To devise an efficient approach for production of human hemangiopoietin (hHAPO), the gene of hHAPO was synthesized and subcloned into the pSUMO vector with a SUMO tag at the N-terminus. The expression construct was then transformed into the expression strain E. coli BL21(DE3). The fusion protein was expressed in soluble form and identified by SDS-PAGE and Western blotting. The fusion protein was purified to 90% purity by metal chelate chromatography with a yield of 45 mg per liter fermentation culture. The SUMO tag was removed by cleavage with SUMO protease at room temperature for 1 h, and the hHAPO was then re-purified by the metal chelate chromatography. Finally, about 21 mg hHAPO was obtained from 1 liter of fermentation culture with no less than 95% purity. The recombinant hHAPO significantly stimulated the proliferation of human umbilical vein endothelial cells.

MeSH terms

  • Blotting, Western
  • Cell Line
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / metabolism*
  • Humans
  • Proteoglycans / genetics
  • Proteoglycans / isolation & purification
  • Proteoglycans / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism

Substances

  • PRG4 protein, human
  • Proteoglycans
  • Recombinant Fusion Proteins