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| Expression and Fusion Protein TAT-NLS-Nkx6.2 in E.coli and Its Purification and Biological Analysis |
| SUN Shao-fei, WANG Bei-lei, YUAN Ting, ZHANG Bing, GUO Gang, ZHANG Ru |
| Institute of Endocrinology/Metabolic Disease Hospital of Tianjin Medical University, Tianjin 300070, China |
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Abstract Objective:To obtain a fusion protein TN-Nkx6.2, which Nkx6.2 links with TAT and NLS in a recombinant prokaryotic system and to analysis its bioactivity. Methods:In this research, we engineer human immunodeficiency virus HIV-1 trans-activating factor TAT and nuclear location sequence (NLS) into transcription factor Nkx6.2 protein ligate to synthetic gene TAT-NLS-Nkx6.2, recombinant plasmid pET-28a- TN-Nkx6.2 and transform into E.coli BL21 (DE3) for expression by IPTG induction.Then by SDS-PAGE and Western Blot to to identified fusion protein,which purified by histidine Ni2+ affinity chromatography.Results:TN-Nkx6.2 was with above 90% purity and good solubility was obtained in vitro,which can combine with brain cells in mice and internalize into the cytoplasm and nucleus proved by fluorescent immunohistochemical.Conclusion:Obtain the fusion protein of TN-Nkx6.2 has biological activity, and can cross the blood-brain barrier into the mice brain cells in nucleus cytoplasm, which lay the material foundation for further explortion of the molecular mechanism that Nkx6.2 hox genes in thyroid reduced model role.
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Received: 03 July 2013
Published: 25 September 2013
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