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Purification and Characterization of Recombinant Darbepoetin Alfa From Leishmania Tarentolae Publisher Pubmed



Kianmehr A1 ; Mahrooz A2 ; Oladnabi M3 ; Safdari Y4 ; Ansari J5 ; Veisi K6 ; Evazalipour M7 ; Shahbazmohammadi H8 ; Omidinia E8
Authors
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Authors Affiliations
  1. 1. Biochemistry & Metabolic Disorders Research Center, Golestan University of Medical Sciences, Gorgan, Iran
  2. 2. Immunogenetic Research Center, Mazandaran University of Medical Sciences, Sari, Iran
  3. 3. Congenital Malformations Research Center, Golestan University of Medical Sciences, Gorgan, Iran
  4. 4. Medical Cellular and Molecular Research Center, Golestan University of Medical Sciences, Gorgan, Iran
  5. 5. Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran
  6. 6. Biotechnology Department, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
  7. 7. Department of Pharmaceutical Biotechnology, School of Pharmacy, Guilan University of Medical Sciences, Rasht, Iran
  8. 8. Enzyme Technology Laboratory, Biochemistry Department, Metabolic and Genetic Research Group, Pasteur Institute of Iran, Tehran, Iran

Source: Molecular Biotechnology Published:2016


Abstract

Darbepoetin alfa is a biopharmaceutical glycoprotein that stimulates erythropoiesis and is used to treat anemia, which associated with renal failure and cancer chemotherapy. We herein describe the structural characterization of recombinant darbepoetin alfa produced by Leishmania tarentolae T7-TR host. The DNA expression cassette was integrated into the L. tarentolae genome through homologous recombination. Transformed clones were selected by antibiotic resistance, diagnostic PCRs, and protein expression analysis. The structure of recombinant darbepoetin alfa was analyzed by isoelectric focusing, ultraviolet–visible spectrum, and circular dichroism (CD) spectroscopy. Expression analysis showed the presence of a protein band at 40 kDa, and its expression level was 51.2 mg/ml of culture medium. Darbepoetin alfa have 5 isoforms with varying degree of sialylation. The UV absorption and CD spectra were analogous to original drug (Aranesp), which confirmed that the produced protein was darbepoetin alfa. Potency test results revealed that the purified protein was biologically active. In brief, the structural and biological characteristics of expressed darbepoetin alfa were very similar to Aranesp which has been normally expressed in CHO. Our data also suggest that produced protein has potential to be developed for clinical use. © 2016, Springer Science+Business Media New York.
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