کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2020327 1542323 2016 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Cloning and expression of codon-optimized recombinant darbepoetin alfa in Leishmania tarentolae T7-TR
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Cloning and expression of codon-optimized recombinant darbepoetin alfa in Leishmania tarentolae T7-TR
چکیده انگلیسی


• A codon-optimized recombinant darbepoetin alfa was expressed in L. tarentolae.
• Expression of secretory protein was calculated to be 11 mg/ml of culture medium.
• The potency of produced darbepoetin alfa was similar to that of Aranesp.
• L. tarentolae can be utilized as an efficient host for darbepoetin alfa production.

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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 118, February 2016, Pages 120–125
نویسندگان
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