Expression and Purification of PhoR Sensor-Domain Histidine Kinase of Mycobacterium tuberculosis in Escherichia coli  

Oleh:

Giri-Rachman, Ernawati Arifin ; Pratama, Fenryco ; Aji, Oktira Roka ; Patriati, Arum ; Ihsanawati ; Putra, Edy Giri Rachman ; Moeis, Maelita Ramdani

Jenis: Article from Journal - ilmiah nasional - terakreditasi DIKTI
Dalam koleksi: Microbiology Indonesia vol. 9 no. 2 (Jun. 2015), page 51-57.
Topik: multi-drug resistance; rational drug design; tuberculosis; two-component signal transduction system of histidine kinase
Fulltext: 321-932_her.pdf (1.14MB)

Isi artikel

Globally, tuberculosis (TB) remains a leading cause of death. The emergence of multidrug-resistant strains (MDR-TB) and extensively drug-resistant strains (XDR-TB) has fuelled the discovery for novel drugs and drug targets for its successful and better treatment. One of the potential candidates for drug target is PhoR sensory protein histidine kinase, a part of the Two Component System (TCS) PhoP/PhoR in Mycobacterium tuberculosis (Mtb). This protein system was known for its role on regulating hundred of Mtb virulence factors, from genes for cell wall and lipid synthesis to genes for adaptation in human leukocyte and hypoxia response. Previous studies have successfully characterized, isolated, and cloned theputative sensory domain of PhoR protein gene into pRSET vector expression system. In this study, Escherichia coli was transformed with pRSET-SensPhoR and cultivated at 37 C under IPTG induction to express PhoR sensor-domain protein. Most of the proteins were overexpressed in the form of inclusion bodies. Subsequent protein purification in Ni-NTA system under refolding condition on urea gradient was performed to isolate PhoR sensor-domain protein in soluble form. Arginine was supplemented in purified protein solution to prevent aggregation during long term storage. While highly purified protein was acquired, small angle X-ray scattering (SAXS) analysis was conducted to obtain 3dimensional (3D) protein structures in solution.

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