title: Cloning and expression of codon-optimized recombinant darbepoetin alfa in Leishmania tarentolae T7-TR.
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.
authors.affiliation: Department of Medical Biotechnology, Faculty of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan, Iran. Electronic address: kiabiotpro@yahoo.com.
authors.forename: Anvarsadat
authors.initials: A
authors.lastname: Kianmehr
authors.affiliation: Department of Genetics, Islamic Azad University, Arsanjan Branch, Fars, Iran.
authors.forename: Raziyeh
authors.initials: R
authors.lastname: Golavar
authors.affiliation: Department of Genetics, Islamic Azad University, Arsanjan Branch, Fars, Iran.
authors.forename: Mandana
authors.initials: M
authors.lastname: Rouintan
authors.affiliation: Immunogenetic Research Center, Mazandaran University of Medical Sciences, Sari, Iran.
authors.forename: Abdolkarim
authors.initials: A
authors.lastname: Mahrooz
authors.affiliation: Department of Biochemistry, Genetics and Metabolism Research Group, Pasteur Institute of Iran, Tehran 13164, Iran.
authors.forename: Pezhman
authors.initials: P
authors.lastname: Fard-Esfahani
authors.affiliation: Department of Medical Genetics, Faculty of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan, Iran.
authors.forename: Morteza
authors.initials: M
authors.lastname: Oladnabi
authors.affiliation: 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.
authors.forename: Safoura
authors.initials: S
authors.lastname: Khajeniazi
authors.affiliation: Department of Genetics, Islamic Azad University, Arsanjan Branch, Fars, Iran. Electronic address: samaneh.mostafavi@gmail.com.
authors.forename: Seyede Samaneh
authors.initials: SS
authors.lastname: Mostafavi
authors.affiliation: Department of Biochemistry, Genetics and Metabolism Research Group, Pasteur Institute of Iran, Tehran 13164, Iran. Electronic address: e.omid8@yahoo.com.
authors.forename: Eskandar
authors.initials: E
authors.lastname: Omidinia
mesh_terms: D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D000068256:Darbepoetin alfa / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D007891:Leishmania / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D003062:Codon; D015800:Protozoan Proteins; D000068256:Darbepoetin alfa
keywords: Codon optimization; Darbepoetin alfa; Expression; Leishmania tarentolae