MUTAGENESIS SITUS TERARAH PLASMID PEMBAWA GEN PENGKODE PROTEIN TATADCD DAN EKSPRESINYA

• Main Author: Claudia, Elisabeth
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/82583
• Institution: Institut Teknologi Bandung

The Twin Arginine Transloca'on (TAT) system in bacteria can facilitate protein transloca'on in a semi-folded or fully folded state. TatAdCd protein transloca'on pathway found in Bacillus sub/lis can be u'lized as a transloca'on pathway for recombinant proteins that have intracellular folding problems in Escherichia coli host cells. In previous research conducted at the ITB Pharmaceu'cal biotechnology laboratory, a frameshi\ muta'on was observed in the codon reading frame of the DNA sequence encoding the TatAd protein. This study aims to eliminate the guanine base causing the muta'on in the DNA sequence encoding the TatAd protein by performing site-directed mutagenesis by Polymerase Chain Reac'on and confirming it at the DNA and protein level. Plasmid carrying the mutated TatAdCd protein-encoding gene was prepared, its muta'on corrected, and then the plasmid was transformed into E. coli DH5a host cells for cloning and confirmed at the DNA level. Plasmid with the corrected DNA sequence was subsequently transformed into the E. coli BL21(DE3) expression host cells. TatAdCd protein was produced on a small scale by IPTG induc'on and confirmed via SDS-PAGE Tris-Tricine. At the DNA level, the plasmid was confirmed through migra'on, restric'on, and nucleo'de sequencing analysis, which showed that the guanine base causing the muta'on had been successfully removed. The SDS PAGE Tris-Tricine analysis revealed the presence of a 27.5 kDa protein band which is presumed to be the TatCd protein. However, the presence of the TatAd protein, which is theore'cally 7.4 kDa, was not observed in this study because proteins smaller than 11 kDa cannot be visualized on the SDS PAGE Tris-Tricine gel.