Purification and Characterization of Recombinant Darbepoetin Alfa from Leishmania tarentolae

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.

Glycoengineering of Darbepoetin-α in CHO-DG44 Cells through Overexpression of α-2,3-sialyl-transferase and CMP-sialic Acid Transporter

“…Those rhEPOs are available in different types which include epoetin-α, epoetin-β or recently darbopoetin-α (Elliott et al 2003). Compared to other EPOs, DPO has two additional N-linked glycosyl groups which enhance its in vivo bioavailability eventhough it has a lower affinity for its receptor in vitro (Kianmehr et al 2016). Thus, the longer halflife of DPO, the future of its clinical application will also be more prospective.…”

“…As glycosylation is important to enhance its bioavailabilty, EPO molecule has been modified to bear 5 N-link glycosyl residues which known as darbepoetin (DPO) (Egrie and Browne 2001;Darling et al 2002;Fan 2015;Falck et al 2017). Containing higher sialic acid contents compared to native EPO (Macdougall 2002;Kianmehr et al 2016), DPO has been proven to have longer in vivo half-life with better efficacy (Egrie et al 2003).…”

Leishmania mexicana recombinant filamentous acid phosphatase as carrier for Toxoplasma gondii surface antigen 1 expression in Leishmania tarentolae

“…Moreover, the selection of recombinant parasites can easily be performed using antibiotic resistance genes, such as phleomycin, which also used in this investigation to generate permanent and stable transfectants, linear DNA for chromosomal integration (Beverley and Clayton 1993 ). However, when using higher DNA amounts homologous integration of multiple copies of the transfected DNA could be observed into the L. tarentolae genome through homologous recombination (Kianmehr et al 2016 ). To confirm integration into the small subunit rRNA gene locus of the L. tarentola e genome by homologous recombination diagnostic PCRs were performed.…”

Leishmania mexicana recombinant filamentous acid phosphatase as carrier for Toxoplasma gondii surface antigen 1 expression in Leishmania tarentolae

“…To generate permanent and stable transfectants, linear DNA has to be used for chromosomal integration (Beverley & Clayton, 1993). However, when using higher DNA amounts homologous integration of multiple copies of the transfected DNA could be observed (Kianmehr et al, 2016). The recombinant DNA cassette is integrated into the L. tarentolae genome through homologous recombination (Kianmehr et al, 2016).…”

“…However, when using higher DNA amounts homologous integration of multiple copies of the transfected DNA could be observed (Kianmehr et al, 2016). The recombinant DNA cassette is integrated into the L. tarentolae genome through homologous recombination (Kianmehr et al, 2016). To con rm integration into the small subunit rRNA gene locus of the L. tarentolae genome by homologous recombination diagnostic PCRs were performed.…”