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Purification and Characterization of Recombinant Darbepoetin Alfa from Leishmania tarentolae

Kianmehr, A. and Mahrooz, A. and Oladnabi, M. and Safdari, Y. and Ansari, J. and Veisi, K. and Evazalipour, M. and Shahbazmohammadi, H. and Omidinia, E. (2016) Purification and Characterization of Recombinant Darbepoetin Alfa from Leishmania tarentolae. Molecular Biotechnology, 58 (8-9). pp. 566-572.

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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.

Item Type: Article
Additional Information: cited By 0
Uncontrolled Keywords: Characterization; Chemotherapy; Circular dichroism spectroscopy; Dichroism; Electrophoresis; Purification, Antibiotic resistance; Biological characteristic; Darbepoetin alfa; Homologous recombination; Isoelectric focusing; Leishmania; Protein expressions; Structural characterization, Proteins
Subjects: مقالات نمایه شده محققین دانشگاه در سایت ,Web of Science ,Scopus
Divisions: معاونت تحقیقات و فناوری
Depositing User: GOUMS
Date Deposited: 07 Sep 2016 09:11
Last Modified: 26 Sep 2016 11:15
URI: http://eprints.goums.ac.ir/id/eprint/4519

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