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

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Abstract

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).

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Abbreviations

PAS:

Proline, alanine, serine

rHuEPO:

Recombinant human erythropoietin

PEG:

Poly ethylene glycol

CD:

Circular dichroism

MTT:

[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]

DMSO:

Dimethyl sulfoxide

t1/2 :

Terminal half-life

RMSD:

Root-mean-square deviation

CZE:

Capillary zone electrophoresis

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Acknowledgements

This research has been supported by Tehran University of Medical Sciences & Health Services Grant 21960. The authors wish to express their deep gratitude to Pasteur Institute of Iran for scientific collaboration during the course of this research.

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Authors and Affiliations

  1. Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran

    Mohammad Hossein Hedayati

  2. Department of Pilot Nanobiotechnology, Pasteur Institute of Iran, Tehran, Iran

    Dariush Norouzian & Reza Ahangari Cohan

  3. Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

    Mahdi Aminian

  4. Department of Medical Genetics, Iran University of Medical Sciences, Tehran, Iran

    Shahram Teimourian

  5. Department of Medical Biotechnology, Pasteur Institute of Iran, Tehran, Iran

    Soroush Sardari

  6. Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, 5th Fl., Dr. Shariati Hospital, North Karegar Ave., Tehran, 1411413137, Iran

    M. Reza Khorramizadeh

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  1. Mohammad Hossein Hedayati
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Correspondence to M. Reza Khorramizadeh.

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There are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. All welfare of animals was conformed to the regulations and guidelines of the National Ethics Committee of the Ministry of Health and Medical Education of Iran.

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Hedayati, M.H., Norouzian, D., Aminian, M. et al. Molecular Design, Expression and Evaluation of PASylated Human Recombinant Erythropoietin with Enhanced Functional Properties. Protein J 36, 36–48 (2017). https://doi.org/10.1007/s10930-017-9699-9

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