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| Prokaryotic Expression of MAP30 from Momordica charantia and Its Biological Activity |
| QIU Hua-li1, RANG Jie1, DING Xue-zhi1, HU Sheng-biao1, ZHANG You-ming1, ZHU Dao-qi2, XIA Li-qiu1 |
1. College of Life Science, Hunan Normal University, Changsha 410081, China;
2. The Second Affiliated Hospital of Hunan Normal University, Changsha 410003, China |
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Abstract Mature map30(signal peptide sequence excluded) was amplified from the genome of bitter melon and inserted on the expression vector pET28a. The final expression vector pET28a-map30 was transformed into E. coli Rostta (DE3) for MAP30 expression. The recombinant MAP30 was identified by SDS-PAGE, Western blotting and LC-MS. After purification by nickel-affinity chromatography, The DNA-cleaving activity was analyzed by electrophoresis after supercoiled plasmid pUC19 as substrate treated with MAP30 of various concentrations. Meanwhile MAP30 was used to treat MCF-7 human breast tumor cells, MTT, DNA Ladder, AO/PI double-stained is used to detect the anti-tumor activity. Results show that map30 gene was successfully expressed in E. coli Rostta (DE3)and been identified by LC-MS and Western blot using His-tag mAb. First discovered through Escherichia coli heterologous expression of recombinant MAP30 like natural MAP30 protein also exert the activity conversion supercoiled plasmid pUC19 to relaxed or linear forms. The cytotoxicity assay of recombinant MAP30 showed that it exhibited dose-dependent and time-dependent inhibition to MCF-7, AO/PI double-stained displayed typical of apoptosis. It through genetic engineering technology preparation MAP30 protein to further study of its biological activities in vitro and laid important foundation for the future clinical application.
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Received: 24 March 2014
Published: 25 June 2014
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