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Protein Expr Purif. 2016 Feb;118:120-5. doi: 10.1016/j.pep.2015.10.013. Epub 2015 Nov 3.

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

Author information

1
Department of Medical Biotechnology, Faculty of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan, Iran. Electronic address: kiabiotpro@yahoo.com.
2
Department of Genetics, Islamic Azad University, Arsanjan Branch, Fars, Iran.
3
Immunogenetic Research Center, Mazandaran University of Medical Sciences, Sari, Iran.
4
Department of Biochemistry, Genetics and Metabolism Research Group, Pasteur Institute of Iran, Tehran 13164, Iran.
5
Department of Medical Genetics, Faculty of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan, Iran.
6
Department of Medical Biotechnology, Faculty of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan, Iran; Stem Cell Research Center, Golestan University of Medical Sciences, Gorgan, Iran.
7
Department of Genetics, Islamic Azad University, Arsanjan Branch, Fars, Iran. Electronic address: samaneh.mostafavi@gmail.com.
8
Department of Biochemistry, Genetics and Metabolism Research Group, Pasteur Institute of Iran, Tehran 13164, Iran. Electronic address: e.omid8@yahoo.com.

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.

KEYWORDS:

Codon optimization; Darbepoetin alfa; Expression; Leishmania tarentolae

PMID:
26546410
DOI:
10.1016/j.pep.2015.10.013
[Indexed for MEDLINE]

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