Cloning of Mycobacteria tuberculosis furA and expression of its coding protein fused with (His)6 tag

    GAO Xue, XUE Ying, JIANG Hong, BAI Yinª²Lan, SHI Changª²Hong, ZHANG Hai, LI Yuan, XU Zhiª²Kai

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

    ¡¾Abstract¡¿ AIM: To obtain furA (ferric uptake regulator A) gene segments of Mycobacteria tuberculosis (MTB), express efficiently in E.coli and purify the target proteins. METHODS: furA gene segments were amplified by PCR with specific primers from genome of MTB H37Rv strain. After sequenced, furA gene segments were inserted into pRSETª²A and expressed in E.coli BL21. The recombinant (His)6 fused proteins were purified by Niª²NTA purification system. RESULTS: The length of PCR product was 453 bp, identical with that reported by Genbank. The proteins were expressed in E.coli BL21 as soluble proteins, accounting for 10% of the total bacterial proteins. SDSª²PAGE and Western blot showed that the Mr of this gene product was 23.0¡Á103. The (His)6 fused proteins were purified by affinity chromatography. CONCLUSION: furA of MTB was successfully cloned and expressed in E.coli BL21. The purified proteins are obtained by Niª²NTA purification system. This work lays the foundation for further study on the ferric uptake of Fe2+ in MTB. ......