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| Preliminary Crystallization of Human Thyroid Hormone Receptor Interacting Protein 15 and Its Expression in Human Tissue |
| WANG Hai-bo1,2, YE Yu-jia1, MENG Zhao-hui1 |
1. Laboratory of Molecular Cardiology, Department of Cardiology, The First Affiliated Hospital of Kunming Medical University, Kunming 650032, China;
2. Biotherapy Center, The General Hospital of Beijing Military Command, Beijing 100700, China |
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Abstract Objective: To construct prokaryotic expression vector of the full length human thyroid hormone receptor interacting protein 15 (hTRIP15), to establish the strategy to express, purify and crystallize hTRIP15; To determinate its expression in human umbilical vein endothelial cells line (HUV-EC-C), platelet, mitral valve, gall bladder and glioma. Methods: Full length coding sequence of hTRIP15 was amplified from human hepatocyte cDNA library by PCR and cloned into pGEX-6P-1 vector after digested with BamH I and Xho I. GST-hTRIP15 was expressed in E.coli BL21 (DE3), and then protein was purified by affinity chromatography and gel filtration chromatography. Hanging drop vapor diffusion method was employed to screen and optimize the crystallizing condition. Crystal structure determination was tried by X-ray crystal diffraction technique. Further, the expression of hTRIP15 in HUE-EC-C, platelet, mitral valve, gall bladder and glioma was evaluated by RT-PCR and Western blot. Results: Recombinant hTRIP15 was successfully expressed in E.coli and purified. The preliminary crystallization condition was determined using the hanging-drop vapor-diffusion method. TRIP15 mRNA was determined by RT-PCR in human HUV-EC-C, platelet, mitral valve, gall bladder and glioma, and its expression in HUV-EC-C was further confirmed by Western blot. Conclusion: The strategy of cloning, expressing and purifying the hTRIP15 was established. The preliminary crystallization condition was obtained. It provides experimental data for final tertiary structure determination. The expression of hTRIP15 was preliminarily determined in HUV-EC-C, platelet, mitral valve, gall bladder and glioma. It provides experiment data for further function study.
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Received: 30 December 2013
Published: 25 April 2014
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