Tropical Journal of Pharmaceutical Research

Original Research Article | OPEN ACCESS

Simple and efficient expression of codon-optimized mouse leukemia inhibitory factor in Escherichia coli

Dewei Niu¹, Qiuhong Wu^1,2, Luyuan Huang³, Enze Yang¹, Meiyan Huang¹, Yong Liu⁴, Feng Wang¹

¹Institute of Genomic Medicine, College of Pharmacy, Jinan University, Guangzhou 510632; ²Department of Pharmacy, Maternal and child health care Hospital of Zaozhuang, Zaozhuang 277100; ³Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530; ⁴Guangdong Key Laboratory of Agro-Environment Integrated Control, Guangdong Institute of Eco-Environmental and Soil Sciences, Guangzhou 510650, China.

For correspondence:- Feng Wang Email: jnubiopharm@126.com Tel:+8615360022165

Received: 9 September 2016 Accepted: 10 March 2017 Published: 30 April 2017

Citation: Niu D, Wu Q, Huang L, Yang E, Huang M, Liu Y, et al. Simple and efficient expression of codon-optimized mouse leukemia inhibitory factor in Escherichia coli. Trop J Pharm Res 2017; 16(4):811-818 doi: 10.4314/tjpr.v16i4.10

© 2017 The authors.
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Abstract

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-mLIF^m 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 rmLIF^m 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 mLIF^m, respectively. His-tagged rmLIF^m 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-speci@257;c 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