Erythropoietin (EPO) is a glycoprotein hormone secreted from primarily cells of the peritubular capillary endothelium of the kidney, and is responsible for the regulation of red blood cell production. We constructed and expressed dimeric cDNAs in Chinease hamster ovary (CHO) cells encoding a fusion protein consisting of 2 complete human EPO domains linked by a 2-amino acid linker (Ile-Asp). We described the activity of dimeric hyperglycosylated EPO (dHGEPO) mutants containing additional oligosaccharide chains and characterized the function of glycosylation. No dimeric proteins with mutation at the 105th amino acid were found in the cell medium. Growth and differentiation of the human EPO-dependent leukemiae cell line (F36E) were used to measure cytokine dependency and in vitro bioactivity of dHGEPO proteins. MTT assay at 24 h increased due to the survival of F36E cells. The dHGEPO protein migrated as a broad band with an average molecular mass of 75 kDa. The mutant, dHGEPO, was slightly higher than the wild-type (WT) dimeri-EPO band. Enzymatic N-deglycosylation resulted in the formation of a narrow band with a molecular mass twice of that of of monomeric EPO digested with an N-glycosylation enzyme. Hematocrit values were remarkably increased in all treatment groups. Pharmacokinetic analysis was also affected when 2.5 IU of dHGEPO were intravenously injected into the tails of the mice. The biological activity and half-life of dHGEPO mutants were enhanced as compared to the corresponding items associated the WT dimeric EPO. These results suggest that recombinant dHGEPO may be attractive biological and therapeutic targets.

ABSTRACT   INTRODUCTION   MATERIALS AND METHODS    Materials    Construction of EPO-EPO Fusion Protein cDNA andSite-directed Mutagenesis    Expression Vector Construction    Cell Culture and Functional Expression    Northern Blot Analysis    Cell Proliferation Assay    SDS-PAGE and Western Blot Analysis    Enzymatic Release of N-Linked Oligosaccharides    In Vivo Biological Activity and Pharmacokinetics Analysis   RESULTS    rec-hEPO Production    Northern Blot Analysis    Cell Proliferation Assay    Western Blot and Treatment of N-linked Oligosaccharide    In Vivo Biological Activity    Pharmacokinetic Analysis   DISCUSSION   REFERENCES

• Purevjargal Naidansuren(Animal Biotechnology, Graduate School of Bio. & Information Technology, Institute of Genetic Engineering, Hankyong National University)
• Kwan-Sik Min(Animal Biotechnology, Graduate School of Bio. & Information Technology, Institute of Genetic Engineering, Hankyong National University) Corresponding author