Document Type : Research articles
Authors
- Behrouz Taheri 1
- Mohammad-Hossein Modarressi 2
- Mahmoud Hashemitabar 3
- Mohammad Miryounesi 4
- Elahe Motevaseli 5
1 Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
3 Department of Anatomical Sciences, Cellular and Molecular Research Center, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
4 Genomic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
5 Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran Food Microbiology Research Center, Tehran University of Medical Sciences, Tehran, Iran
Abstract
Background: Leukemia Inhibitory Factor (LIF) is largely used in stem cell researches for the maintenance of Embryonic Stem Cells (ESCs) in a pluripotent state. However, the relatively high costs of LIF is a potential limit of such researches.
Objectives: The aim of this study was prokaryotic expression and purification of the recombinant hexahistidine-tagged human LIF (His6-hLIF) fusion protein and assessment of its ability to maintain a pluripotent or undifferentiated state of ESCs. Methods: Encoding the DNA sequence of mature hLIF was codon optimized for expression in Escherichia coli and chemically syn- thesized and cloned in the expression vector pET- 28a (+). Immobilized Metal Affinity Chromatography (IMAC) was performed to purify the recombinant His6-hLIF. Then, His6-hLIF was tested for its ability to maintain mESC by comparison with commercial LIF as a control. Results: The yield for the recombinant His6-hLIF was assessed to be approximately 1.7 mg from one liter of culture. There were no statistically significant differences in expression of two pluripotency gene markers, oct-4 and Nanog, between mESCs treated with
His6-hLIF and those with commercial hLIF (P = 0.09 and P = 0.13, respectively). Besides, morphological characteristics (round cellular morphology) were similar between them. Conclusions: Collectively, the findings showed that the ability of the recombinant His6-hLIF protein in maintaining pluripotent state of ESCs was comparable to commercial hLIF, providing evidence that the presence of the N-terminal hexahistidin tag does not influence biological activities of hLIF.
Keywords
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