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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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Kianmehr A,Golavar R,Rouintan M,Mahrooz A,Fard-Esfahani P,Oladnabi M,Khajeniazi S,Mostafavi SS,Omidinia E

doi

10.1016/j.pep.2015.10.013

subject

Has Abstract

pub_date

2016-02-01 00:00:00

pages

120-5

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(15)30092-9

journal_volume

118

pub_type

杂志文章

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