Prokaryotic expression and purification of recombinant human leukemia inhibitory factor : analysis of the ability to maintain pluripotency in embryonic stem cells

الملخص EN

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.