THE DEVELOPMENT OF A NOVEL SCREENING SYSTEM FOR NEW ANTITUBERCULAR IN Escherichia coli BL21(DE3)pLysS BASED ON FUSION OF PHOR CYTOPLASMIC DOMAIN OF Mycobacterium tuberculosis H37Rv AND ICLR REPRESSOR

• Main Author: Sopiyani K, Irma
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/34982
• Institution: Institut Teknologi Bandung

The emergence of Multi-Drug and Extensively-Drug Resistant Tuberculosis (MDR and XDR-TB) has revealed the need for the discovery of novel antitubercular drugs. The characteristic of current antitubercular drugs basically one drug which just targeting one gene product, hence we are trying to investigate a drug target which could influence a number of genes in Mycobacterium tuberculosis which are known to have a role in virulence. One of the potential candidate as targets of new antitubercular drug development is the cytoplasmic domain of protein PhoR histidine kinase (CytophoR) conducted through the high throughput screening system based on dimerization inhibition of CytophoR. This system utilizes the dimerization properties of IclR repressor protein from Escherichia coli which will then be used as a tool to detect the CytophoR dimerization through the fusion of DNA binding domain (100N-terminal) of IclR repressor protein and CytophoR. The presence of inhibitors that can prevent dimerization of the IclR-CytophoR fusion protein would result in IclR which could bind to the repressor binding site of the the iclR promoter that was located the upstream of the reporter gene emerald green fluorescent protein (emgfp). Dimerization and its inhibition would be confirmed through EmGFP expression. CytophoR gene was succesfully characterized, isolated from Mtb H37Rv and fused with the DNA binding domain of IclR repressor. The results showed that CytophoR was located at amino acid 177-436 from all PhoR intact and characterized by conserved motifs H-box, N-box, G1-box, F-box, and G2-box. CytophoR gene was fused with the iclR forming iclR-cytophoR fusion gene and then constructed into the pRSET-promiclR expression vector. IclR-CytophoR fusion protein was successfully expressed in the E. coli BL21(DE3)pLysS by 1 mM IPTG induction for 4 h and then analyzed by SDS PAGE. The results showed a protein band with a size of ~36 kDa which was predicted to be the monomer of IclR-CytophoR. The dimerization of IclR-CytophoR fusion protein would be expected to be the target for new antitubercular screening system. This system would select drug candidates which would successfully inhibit the dimerization process of IclR-sitophoR fusion protein.