Simple and efficient expression of codon-optimized mouse leukemia inhibitory factor in Escherichia coli
Purpose: To obtain a higher yield of mouse leukemia inhibitory factor to maintain the proliferation potential of pluripotent stem cells at a low cost.
Methods: A method was designed to produce recombinant mLIF protein (rmLIF) in Escherichia coli. Through analysis of rmLIF sequence, it was found that rare codons were interspersed. After mutation from rare codons to Escherichia coli (E. coli) preferred ones were selected, the mutated gene mLIFm was cloned into pET15b vector. The pET15b-mLIFm was then transformed into Rosetta-gami strain and induced with optimal conditions at 18 oC for 16 h. Mass spectrometry was carried out to identify the peptides.
Results: After purification, the yield of the codon-optimized rmLIFm was 141 mg/L, compared with 110 mg/L for the original rmLIF. Mass spectral analysis showed the presence of four major peptides each with an intensity > 10 % at m/z 1031.57, 1539.82, 1412.01 and 2229.10 in mLIFm, respectively. Histagged rmLIFm fusion protein displayed the potential to maintain the morphology of undifferentiated mouse embryonic stem cells (mESCs), which were positive for mESCs markers (Oct-4, Nanog, Sox-2, stage-specific embryonic antigen-1).
Conclusion: The findings provide a means to produce mLIF in a short, useful, cost-effective and environmentally-friendly manner, and thus lays a foundation for further studies of mLIF.
Keywords: Leukemia inhibitory factor, Mutated gene, Protein expression, Purification, Stem cells, Peptides, Escherichia coli
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