Molecular Design, Expression and Evaluation of PASylated Human Recombinant Erythropoietin with Enhanced Functional Properties

Protein J. 2017 Feb;36(1):36-48. doi: 10.1007/s10930-017-9699-9.


Erythropoietin (EPO) is the principal hormone which, has somewhat short half-life involved in the differentiation and regulation of circulating red blood cells. The present study was carried out to evaluate the capability of a polyethylene glycol mimetic technology as a biological alternative to improve pharmaceutical properties of human recombinant EPO. In silico models of EPO fused to 200 amino acids of proline, alanine, and serine (PAS) were initially generated and assessed by molecular dynamic (MD) simulation. The fluctuations of the modeled structure reached a plateau after 6000 ps of MD simulation. The Phi and psi analysis showed >99.2% of residues were located in the allowed regions. An expression vector consisting of EPO cDNA tagged to PAS coding sequences was synthesized and expressed in CHO-K1 Cells. The produced PASylated molecule was purified and characterized by standard analytical methods. The molecular weight of fusion protein was expanded to 70 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis method. Analytical size exclusion chromatography revealed an approximately sevenfold increase in apparent size of produced protein. Although the in vitro potency of the fusion protein was significantly reduced (1.26 ± 0.05 vs. 0.24 ± 0.03 ng/ml) but, the in vivo activity was considerably increased up to 1.58 × 105 IU/ml in normocythemic mice assay. Pharmacokinetic animal studies revealed strongly 15.6-fold plasma half-life extension for the PASylated EPO (83.16 ± 13.28 h) in comparison to epoetin α (8.5 ± 2.4 h) and darbepoetin α (25.3 ± 2.2h).

Keywords: Biological activity; Erythropoietin; Molecular dynamic; PASylation; Pharmacokinetic properties; Plasma half-life.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohol Oxidoreductases
  • Animals
  • Cricetinae
  • Erythropoietin* / chemistry
  • Erythropoietin* / genetics
  • Erythropoietin* / pharmacokinetics
  • Erythropoietin* / pharmacology
  • Gene Expression
  • Humans
  • Mice
  • Molecular Dynamics Simulation*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / pharmacokinetics
  • Recombinant Proteins / pharmacology


  • EPO protein, human
  • Recombinant Proteins
  • Erythropoietin
  • Alcohol Oxidoreductases
  • CHO aldo-keto reductase