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Purification and Characterization of Recombinant Darbepoetin Alfa from Leishmania tarentolae

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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.

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Acknowledgments

This project was financially supported by Department of Research & Technology, Golestan University of Medical Sciences, Gorgan, Iran. The authors also wish to express their deep gratitude to all who provided support, especially Enzyme technology lab., Metabolic and Genetic Research Group, Pasteur Institute of Iran.

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Correspondence to Hamid Shahbazmohammadi or Eskandar Omidinia.

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This article does not contain any studies with human participants performed by any of the authors.

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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

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Anvarsadat Kianmehr and Abdolkarim Mahrooz contributed equally to this work.

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Kianmehr, A., Mahrooz, A., Oladnabi, M. et al. Purification and Characterization of Recombinant Darbepoetin Alfa from Leishmania tarentolae . Mol Biotechnol 58, 566–572 (2016). https://doi.org/10.1007/s12033-016-9954-x

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