Journal of Glycobiology
Open Access

Production of hyper glycosylated darbepoetin alfa using Leishmania tarentolae

Glycobiology World Congress

August 10-12, 2015 Philadelphia, USA

Anvarsadat Kianmehr

Posters-Accepted Abstracts: J Glycobiol

Abstract :

Darbepoetin alfa known by the trade name of Aranesp is a hyper glycosylated and synthetic analog of erythropoietin (EPO)
which is used as a drug in treating anemia in patients with chronic kidney failure and cancer. This study describes the
expression of a codon-optimized recombinant darbepoetin alfa in Leishmaina tarentolae T7-TR host. The gene of darbepoetin
alfa was optimized according to the codon usage of L. tarentolae and synthesized. The synthetic codon optimized gene was
amplified by PCR and cloned into the pLEXSY vector. The resultant expression construct (pLEXSYDrabo) was purified,
digested with SwaI enzyme and electroporated into L. tarentolae. The homologous recombination integration into the L.
tarentolae genome was confirmed by different diagnostic PCRs. Expression of recombinant darbepoetin alfa was analyzed by
ELISA, reverse-transcription PCR (RT-PCR) and Western blotting. The recombinant protein was purified and its biological
activity was also measured.
After codon optimization, Codon Adaptation Index (CAI) was raised from 0.50 to 0.99 and GC% content was changed from 56%
to 58%. Expression analysis showed and confirmed the presence of a protein band at about 40 kDa and its expression level was 11 mg/
mL of culture medium. Reticulocyte experiment results revealed that the activity of expressed darbepoetin alfa was similar with that
of Aranesp. In conclusion, we generated a recombinant L. tarentolae strain expressing the codon-optimized darbepoetin alfa gene.