CONSTRUCTION AND EXPRESSION OF KatG Mutant WITHOUT 62 C-TERMINAL AMINO ACID RESIDUES IN Eschericia coli

• Main Author: DEVI ANGGRAINI (NIM : 10508014);, IRIKA
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/16101
• Institution: Institut Teknologi Bandung

Catalase-peroxidase (KatG) is a bifunctional homodimer enzyme which plays a role in the virulence of Mycobacterium tuberculosis by its catalytic activity in decomposition of <br /> <br /> <br /> <br /> <br /> oxidative compound. KatG has an intracellular activity to convert INH as a prodrug into isonicotinoil-NAD, an inhibitor of mycolic acid biosynthetis which is a primary component of Mycobacterium tuberculosis cell wall and hence katG mutation could lead a phenotype of Isoniazid (INH) resistance Mycobacterium tuberculosis. KatG has two domains namely a Nterminal domain containing active site of heme and a C-terminal domain, which has unknown function yet. Deletion of C-terminal domain is predicted to affect the stability of KatG. In this study, 62 C-terminal amino acid residues of KatG were deleted as an attempt to study its function. The research works included amplification of katG-deltaC62 DNA fragment by PCR, cloning of PCR-amplified product of katG-deltaC62 fragment into pGEM-T vector, followed by sub cloning into pCold expression vector, and katG-deltaC62 expression in E. coli BL21(DE3). Restriction, PCR, and sequence analysis revealed that recombinant vector of pGEM-T-katG-delta C62 and pCold-katG-deltaC62 have been successfully constructed. SDSPAGE analysis showed that katG-deltaC62 has been expressed in E. coli BL21(DE3) as <br /> <br /> <br /> <br /> <br /> inclusion bodies with molecular weight 73,18 kDa theoritically. Model structure of katG-deltaC62 indicates that the 62 C-terminal amino acid residues might have a role in the stability of KatG through nonbonding interaction in dimer interface.