Prokaryotic Expression and Purification of Recombinant Human Leukemia Inhibitory Factor; Analysis of the Ability to Maintain Pluripotency in Embryonic Stem Cells Publisher



Taheri B¹ ; Modarressi MH^{2, 3} ; Hashemitabar M⁴ ; Miryounesi M⁵ ; Motevaseli E^{2, 6}
Source: Iranian Red Crescent Medical Journal Published:2018

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 synthesized 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. © 2018, Author(s).