Mycobacterium tuberculosis RpfA protein expression purification and identification of.doc

 PAGE \* MERGEFORMAT 14 Mycobacterium tuberculosis RpfA protein expression purification and identification of Author: Gao Hui, Bai Yin-lan, Li-Mei Wang, Zhi-Kai Xu, Ying Xue [Keywords:] RpfA; expression; purification; Mycobacterium tuberculosis Expression, purification and identification of Mycobacterium tuberculosis RpfA [Abstract] AIM: To express efficiently Mycobacterium tuberculosis RpfA in E. coli and purify the fusion proteins. METHODS: RpfA gene segments were digested with NdeI and BamHI and cloned into pcDNA3.1 vector. After sequencing, RpfA were subcloned into the prokaryotic expression vector pET19b to get pET19bRpfA. The plasmid pET19bRpfA were transformed into E. coli DE3 and induced by IPTG for its expression. The RpfA fusion protein expression was analyzed by SDSPAGE and confirmed by Western blot. Recombinant (His) 6 fusion proteins were purified via Ni2 NTA affinity chromatography. RESULTS: RpfA gene segments were identical to that GenBank reported (lacking 5 ‘ends of 99 bp). The pET19bRpfA vector expressed RpfA fusion proteins at Mr about 80 ku, which could be caught by anti (His) 6 mAb, antiRv1884 and antiRv2389 immune serum.SDSPAGE analysis showed that the fusion proteins mainly existed in inclusion bodies.The expressed proteins could be purified via Ni2 NTA affinity chromatography in denatured condition. CONCLUSION: The recombinant expression plasmid pET19bRpfA has been constructed and RpfA fusion proteins been successfully expressed in E. coli and purified, which lay a basis for further study of RpfA functions. [Keywords] RpfA; expression; purification; Mycobacterium tuberculosis [Abstract] Objective: Expression and purification of fusion proteins RpfA. Methods: RpfA gene fragment containing the plasmid with NdeI and BamH Ⅰ restriction enzyme digestion, and then the target gene fragment was cloned into the vector pcDNA3.1 recombinant vector pcDNA3.1 RpfA, sequencing, and then the target gene fragments were subcloned into the pET19