Cloning, Overexpression, and Purification of PhoR Cytoplasmic Domain Protein from Mycobacterium tuberculosis strain H37Rv  

Oleh:

Aji, Oktira Roka ; Pascapurnama, Dyshelly Nurkartika ; Pratama, Fenryco ; Ihsanawati ; Moeis, Maelita Ramdani ; Giri-Rachman, Ernawati Arifin

Jenis: Article from Journal - ilmiah nasional - terakreditasi DIKTI
Dalam koleksi: Microbiology Indonesia vol. 08 no. 4 (Dec. 2014), page 141-146.
Topik: Mycobacterium tuberculosis; rational drug design; tuberculosis; two-component system
Fulltext: 284-866-1-PB_Ros.pdf (740.4KB)

Isi artikel

Tuberculosis is still a major health problem in the world. This infectious disease is caused by Mycobacterium tuberculosis (Mtb). Novel anti-tubercular drug is urgently needed to counter multidrug resistant cases and Mtb's spread. The cytoplasmic domain of PhoR histidine kinase, a part of the two-component system PhoR-PhoP in Mtb, is one of the potential candidates for anti-tubercular drug target. Three dimensional structure (3D-structure) of the protein (drug target) is needed to screen potential drug candidate using rational drug design approaches. Previous studies have successfully characterized and isolated putative cytoplasmic domain of PhoR (CytoPhoR) from Mtb strain H37Rv. This study aimed to clone, overexpress, and purify CytoPhoR protein. CytoPhoR was fused with thioredoxin protein in pET32b expression vector and overexpressed in Escherichia coli (E.coli) BL21(DE3) as soluble fraction by induction with 1 mM IPTG. Purification of His-tagged CytoPhoR was carried out using IMAC Ni-NTA Agarose His-tag affinity column. SDS-PAGE analysis showed that another protein was co-purified (~35 kDa) along with the CytoPhoR protein. Subsequent protein purification using DEAE-ion exchange column generated a strong single band of 37 kDa on SDS–PAGE which was identified as CytoPhoR protein. The purified CytoPhoR protein was successfully obtained and could be used for further analysis to determine the 3D-structure of CytoPhoR protein.

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