Isolation and Purification of Recombinant Sensor-Domain PhoR from Mycobacterium tuberculosis

• Main Author: NUR’AINI (NIM : 10513007), DERIN
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/26504
• Institution: Institut Teknologi Bandung

<p align="justify">Tuberculosis is a deadly disease caused by a pathogenic bacterium called Mycobacterium tuberculosis (M. tuberculosis). The increasing number of tuberculosis cases on the last decade has been linked to unclear pathogenesis mechanisms of M. tuberculosis. The appearance of new Mycobacterium tuberculosis galurs which are resistant to anti tubercular drugs are also believed to play a role at latter case. One of the target proteins for studying the pathogenic mechanism of M. tuberculosis is the PhoR-PhoP system, it is a two-component signaling system (TCS) for M. tuberculosis, which is not present in eukaryotes (mammals). This system can also be a drug target in order to cure tuberculosis. PhoR proteins act as signal sensors to activate the virulence genes of M. tuberculosis. Structural knowledge is required to study the interaction of PhoR with its ligands, so that the anti tubercular drugs candidate in the form of a PhoR inhibitor can be determined.. To achieve this goal, the PhoR coding gene in the pRSET-emGFP expression vector is expressed optimally so that PhoR protein can be produced in large scale, soluble in bufer, and has high purity. In order to do that, PhoR gene in recombinant plasmid pRSET-emGFP-PhoR was expressed using 0.1 mM Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) at 20oC temperature for 16 hour. The results of gene expression can be known using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE electrophoregram of PhoR showed a significant band at ~17 kDa of size, which suits with PhoR size by deducing from its nucleotide sequence. But, the produced PhoR formed inclusion bodies (precipitation). Thus, it was needed to do PhoR refolding in order to obtain bufer-soluble protein. PhoR precipitation was denaturized with bufer containing 8 M urea, and then refolded by decreasing urea concentration until 0 M. The refolding result was purified with nikel-nitroloacetic acid (Ni-NTA) affinity chromatography. The PhoR proteins bind to the Ni-NTA matrix are then eluted with a buffer containing 50 &#822; 500 mM imidazole gradient. SDS-PAGE electrophoregram analysis showed that PhoR was successfully purified by bufer containing 250 mM imidazole. The pure PhoR protein was indicated by a single band on SDS-PAGE electrophoregram, with its concentration of 1,28 mg/mL. The truth of pure ds-PhoR protein is validated by mass spectroscopy techniques.<p align="justify">