Endah Puji Septisetyani, Yana Rubiyana, Popi Hadi Wisnuwardhani, Andri Wardiana, Adi Santoso


Stability  of  erythropoietin  (EPO)  depends  on  its glycosylation  states.  With  more  glycosylation  sites,  the  EPO protein  will  be  more  stable  and  also  increase  its  half-life.  A construct  of  recombinant  human  erythropoietin  (rhEPO)  which contains 2 additional N-link for glycosylation were designed. Based on translation analysis using ORF (open reading frame)-finder and protein  alignment  analysis  using  blast-p  of  NCBI  home  page, expected  recombinant  hEPO  with  additional  6-histidin  tag  in carboxyl terminus  was expressed. HEK293T cells  were transfected with  recombinant  plasmid  containing  rhEPO  by  using  calcium phosphate method. Expression of rhEPO was detected by dot blot and  Western  blot  analysis  using  hEPO  antibody  as  the  primary antibody  and  antirabbit  antibody  with  alkaline  phospatase  linked as  the  secondary  antibody.  The  bands  were  detected  by BCIP/NBT color  development  substrate.  The  data  indicated detection of EPO in culture medium of transfected HEK293T cells.

Key  words:  HEK293T  cell,    calcium    phosphate    transfection,  N-linked glycosylation, recombinant human erythropoietin

Full Text:



Ashley, R. A., Dubuque, S. H., Dvorak, B., Woodward, S. S., Williams, S. K., and Kling, P. J., 2002, Erythropoietin Stimulates Vasculogenesis in Neonatal Rat Mesenteric Microvascular Endothelial Cell, Pedriatic Res., 51,471-478.

Backliwal, G., Hildinger, M., Chenuet, S., Wulhfard, S., Jesus, M. D., and Wurm, F. M., 2008, Rational Vector Design and Multi-pathway Modulation of HEK 293E Cells Yield Recombinant Antibody Titers Exceeding 1g/l by Transient Transfection Under Serum-Free Condition, Nuc. Ac. Res., 36(15), e96.

Bollin, F., Dechavanne, V., and Chevalet, L., 2011, Design Experiment in CHO and HEK Transient Transfection Condition Optimization, Protein Exp. and Purif., 78, 61-68.

Cereghino, G. P. L. and Cregg, J. M., 2000, Heterologous Protein Expression in the Methylotrophic Yeast Pichia pastoris, FEMS Microbiol. Rev., 24, 45-66.

Dasgupta, S., Navarrete, A. M., Bayry, J., Delignat, S., Wootla, B., Andre, S., Christope O., Nascimbeni, M., Jacquemin, M., Martinez-Pomares, L., et al., 2007, A Role for Exposed Mannosylations in Presentation of Human Therapeutic Self-Proteins to CD4+ T Lymphocytes. Proc. Natl. Acad. Sci. USA, 104, 8965-8970.

Durocher, Y. and Butler, M., 2009, Expression Systems for Therapeutic Glycoprotein Production, Current Opinion in Biotechnology, 20, 700-707.

Egrie, J. C. and Browne, J. K., 2001, Development and Characterization of Novel Erythropoiesis Stimulating Protein (NESP), Nephrol. Dial. Transplant., 16[Suppl 3], 3-13.

Fischer, R., Drossard, J., Emans, N., Commandeur, U. and Hellwig, S., 1999, Towards Molecular Farming in the Future: Pichia pastoris-Based Production of Single-Chain Antibody Fragments, Biotechnol. Appl. Biochem., 30, 117-120.

Goldwasser, E., 1975, Erythropoietin and the Differentiation of Red Blood Cells, Fed. Proc., 34(13), 2285-2292.

Gross, A.W. and Lodish, H.F., 2006, Cellular Trafficking and Degradation of Erythropoietin and Novel Erythropoiesis Stimulating Protein (NESP), J. Bio. Chem., 281(4), 2024-2032.

Helena, D. A., Jyothsna, N. L., Rao, P. J., Rao, G. V., and Rao, K. R. S. S., 2006, Therapeutic Implications of Recombinant Human Erythropoietin in Anaemic Related Clinical Manifestations, African J. Biotechnology, 5(25), 2503-2509.

Jordan, M., Schallhorn, A., and Wurm, F. M., 1996, Transfecting Mammalian Cells: Optimization of Critical Parameter Affecting Calcium-Phosphate Precipitate Formation. Nuc. Ac. Res., 24(4), 596-601.

Koury, M. J., Bondurant, M. C., 1991, The Mechanism of Erythropoietin Action, Am. J. Kidney Dis., 18(4Suppl1), 20-23.

Lam, J.S., Huang, H., Levitz, S.M., 2007, Effect of Differential N-linked and O-linked Mannosylation on Recognition of Fungal Antigents by Dendritic Cells. PLoS ONE, 2, e1009.

Loignon, M., Perret, S., Kelly, J., Boulais, D., Cass, B., Bisson, L., Afkhamizarreh, F., and Durocher, Y., 2008, Stable High Volumetric Production of Glycosylated Human Recombinant IFNalpha2b in HEK293 Cells, BMC Biotechnology, 8, 65.

Lotscher, N. G., 2005, Anaemia Management in Dialysis Patients in Switzerland “AIMS”, Dissertation, Universitat Basel, Switzerland.

Mariati, Ho, S. C. L., Yap, M. G. S., and Yang, Y., 2010, Evaluating PostTranscriptional Regulatory Elements for Enhancing Transient Gene Expression Levels in CHO K1 and HEK293 Cells, Protein Expr. Purif., 69, 9-15.

Moore K.L., 2003, The Biology and Enzymology of Protein Tyrosine Osulfation. J. Biol. Chem., 278, 24243-24246.

Narhi, L. O., Arakawa, T. Araki, K. H., Elmore, R., Rohde, M. F., Boone, T., and Strickland, T. W., 1991, The Effect of Carbohydrate on The Structure and Stability of Erythropoietin, The Journal of

Biol. Chem., 266(34), 23022-23026. Patell, V. M. U. S., Patent No. 2009/0029907 A1, 29 Jan. 2009.

Sola, R. J. and Griebenow, K., 2009, Effects of Glycosylation on The Stability of Protein Pharmaceuticals, J. Pharm. Sci., 98(4), 1223-1245.

Sola, R. J. and Griebenow, K., 2010, Glycosylation of Therapeutic Proteins: An Effective Strategy to Optimize Efficacy, BioDrugs, 24(1), 9-21.Strickland T. W., U.S. Patent No. 5856298, 5. Jan. 1999.

Suzuki, J., Fukuda, M., Kawata, S., Maruoka, M., Kubo, Y., Takeya, T., and Shishido, T., 2006, A Rapid Protein Expression and Purification System Using Chinese Hamster Ovary Cells Expressing

Retrovirus Receptor, J. Biotec.126, 463-474.

Wardiana, A., and Santoso, A., 2011, Purification and Carbohydrate Analysis of Recombinant Human Erythropoietin Expressed in Yeast System Pichia pastoris. MAKARA SAINS, 15(1), 75-78.

Yoon S. K., Ahn Y. and Han K., 2001, Enhancement of Recombinant Erythropoietin Production in CHO Cells in an Incubator without CO2 Addition, Cytotechnology, 37, 119-132.



  • There are currently no refbacks.


Creative Commons License
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.

Indonesian J Pharm indexed by:

analytics View My Stats