Expression, purification and identification of Mycobacterium tuberculosis RpfA

    GAO Hui, BAI Yinª²Lan, WANG Liª²Mei, XU Zhiª²Kai, XUE Ying

    Department of Microbiology, School of Basic Medicine, Fourth Military Medical University, Xi¬ðan 710033, China

    ¡¾Abstract¡¿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 pET19bª²RpfA. The plasmid pET19bª²RpfA were transformed into E. coli DE3 and induced by IPTG for its expression. The RpfA fusion protein expression was analyzed by SDSª²PAGE and confirmed by Western blot. Recombinant (His)6 fusion proteins were purified via Ni2+ª²NTA affinity chromatography. RESULTS: RpfA gene segments were identical to that GenBank reported (lacking 5¡ä ends of 99 bp). The pET19bª²RpfA vector expressed RpfA fusion proteins at Mr about 80 ku, which could be caught by antiª²(His)6 mAb ,antiª²Rv1884 and antiª²Rv2389 immune serum.SDSª²PAGE analysis showed that the fusion proteins mainly existed in inclusion bodies.The expressed proteins could be purified via Ni2+ª²NTA affinity chromatography in denatured condition. CONCLUSION: The recombinant expression plasmid pET19bª²RpfA 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. ......