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Cloning and expression of codon-optimized recombinant darbepoetin alfa in Leishmania tarentolae T7-TR

Kianmehr, A. and Golavar, R. and Rouintan, M. and Mahrooz, A. and Fard-Esfahani, P. and Oladnabi, M. and Khajeniazi, S. and Mostafavi, S.S. and Omidinia, E. (2016) Cloning and expression of codon-optimized recombinant darbepoetin alfa in Leishmania tarentolae T7-TR. Protein Expression and Purification, 118. pp. 120-125.

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Abstract

Darbepoetin alfa is an engineered and hyperglycosylated analog of recombinant human erythropoietin (EPO) which is used as a drug in treating anemia in patients with chronic kidney failure and cancer. This study desribes the secretory expression of a codon-optimized recombinant form of darbepoetin alfa in Leishmania tarentolae T7-TR. Synthetic codon-optimized gene was amplified by PCR and cloned into the pLEXSY-I-blecherry3 vector. The resultant expression vector, pLEXSYDarbo, was purified, digested, and electroporated into the L. tarentolae. Expression of recombinant darbepoetin alfa was evaluated by ELISA, reverse-transcription PCR (RT-PCR), Western blotting, and biological activity. After codon optimization, codon adaptation index (CAI) of the gene raised from 0.50 to 0.99 and its GC content changed from 56 to 58. Expression analysis confirmed the presence of a protein band at 40 kDa. Furthermore, reticulocyte experiment results revealed that the activity of expressed darbepoetin alfa was similar to that of its equivalent expressed in Chinese hamster ovary (CHO) cells. These data suggested that the codon optimization and expression in L. tarentolae host provided an efficient approach for high level expression of darbepoetin alfa. © 2015 Elsevier Ltd. All rights reserved.

Item Type: Article
Additional Information: cited By 1
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 08:33
Last Modified: 13 Sep 2016 08:44
URI: http://eprints.goums.ac.ir/id/eprint/4586

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