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| Expression, Purification and Bioactivity Identification of Recombinant Human Leukemia Inhibitory Factor (hLIF) Fusion Protein |
| SUN Jing, WANG Bin, DUAN Zhi-qing, HU Ning-zhu, LI Jian-fang, LI Yan-han, HU Yun-zhang |
| Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming 650118, China |
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Abstract Objective: To express recombinant human Leukemia inhibitory factor (hLIF) fusion protein in prokaryotic cells, purify and identify the bioactivity of expressed product. Methods: The hLIF gene was cloned into vector pThioHisA, and the constructed recombinant plasmid pThioHisA-hLIF was transformed to E.coli BL21(DE3) for expression under induction of IPTG. After the expression product waspurified by affinity chromatography, the fusion protein was tested for its specificity by western blot detection. To assess the bioactivity of the hLIF fusion protein expressed in E.coli, it was tested to maintain the pluripotency of the mouse embryonic stem cells in feeder-independent culture system. Results: Lower the induction temperature and prolong the induction time can increase soluble hLIF fusion protein expression. The recombinant protein reached a purity of more than 95% after purification, and its specificity was successfully proved by western blot detection. In feeder-independent culture system, adding hLIF fusion protein can effectively maintain the mouse embryonic stem cells in an undifferentiated state. Conclusion: The recombinant hLIF fusion protein was highly expressed in E.coli and showed good specificity, which lay the foundation for stem cells research and further research on hLIF biological functions.
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Received: 15 January 2013
Published: 25 May 2013
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