Purification and Characterization of Recombinant Darbepoetin Alfa from Leishmania tarentolae.
Darbepoetin alfa is a biopharmaceutical glycoprotein that stimulates erythropoiesis and is used to treat anemia, which associated with renal failure and cancer chemotherapy. We herein describe the structural characterization of recombinant darbepoetin alfa produced by Leishmania tarentolae T7-TR host. The DNA expression cassette was integrated into the L. tarentolae genome through homologous recombination. Transformed clones were selected by antibiotic resistance, diagnostic PCRs, and protein expression analysis. The structure of recombinant darbepoetin alfa was analyzed by isoelectric focusing, ultraviolet-visible spectrum, and circular dichroism (CD) spectroscopy. Expression analysis showed the presence of a protein band at 40 kDa, and its expression level was 51.2 mg/ml of culture medium. Darbepoetin alfa have 5 isoforms with varying degree of sialylation. The UV absorption and CD spectra were analogous to original drug (Aranesp), which confirmed that the produced protein was darbepoetin alfa. Potency test results revealed that the purified protein was biologically active. In brief, the structural and biological characteristics of expressed darbepoetin alfa were very similar to Aranesp which has been normally expressed in CHO. Our data also suggest that produced protein has potential to be developed for clinical use.
Biochemistry & Metabolic Disorders Research Center, Golestan University of Medical Sciences, Gorgan, Iran.
Anvarsadat
A
Kianmehr
Immunogenetic Research Center, Mazandaran University of Medical Sciences, Sari, Iran.
Abdolkarim
A
Mahrooz
Congenital Malformations Research Center, Golestan University of Medical Sciences, Gorgan, Iran.
Morteza
M
Oladnabi
Medical Cellular and Molecular Research Center, Golestan University of Medical Sciences, Gorgan, Iran.
Yaghoub
Y
Safdari
Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran.
Javad
J
Ansari
Biotechnology Department, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Kamal
K
Veisi
Department of Pharmaceutical Biotechnology, School of Pharmacy, Guilan University of Medical Sciences, Rasht, Iran.
Mehdi
M
Evazalipour
Enzyme Technology Laboratory, Biochemistry Department, Metabolic and Genetic Research Group, Pasteur Institute of Iran, Tehran, Iran. hamidbiozyme@gmail.com.
Hamid
H
Shahbazmohammadi
Enzyme Technology Laboratory, Biochemistry Department, Metabolic and Genetic Research Group, Pasteur Institute of Iran, Tehran, Iran. eomid8@yahoo.com.
Eskandar
E
Omidinia
D002942:Circular Dichroism; D003001:Cloning, Molecular; D000068256:Darbepoetin alfa / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D007891:Leishmania / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D020033:Protein Isoforms / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D020033:Protein Isoforms; D011994:Recombinant Proteins; D000068256:Darbepoetin alfa
Characterization; Darbepoetin alfa; Leishmania tarentolae; Purification