#TITLE_ALTERNATIVE#

• Main Author: IMANSARI NIM: 20715305, MUTIARA
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/29427
• Institution: Institut Teknologi Bandung

The p53 protein is a regulatory protein that can inhibit tumor formation by <br /> <br /> activating cell cycle arrest or apoptosis in cell repair systems. It can interact with <br /> <br /> the hepatitis B virus X protein (HBx) which is often associated as a cause of liver <br /> <br /> cancer. The aims of this study are to overproduce and characterize human p53 <br /> <br /> protein with and without coexpression of GroEl-DnaK chaperones on Escherichia <br /> <br /> coli BL21(DE3). The optimization of p53 protein coding gene sequence was <br /> <br /> performed to obtain Codon Adaptation Index (CAI) and %GC of 0.724 and 52.5% <br /> <br /> respectively, which approached the parameters for E. coli B. The optimized p53 <br /> <br /> sequence was inserted on a pET-16b plasmid (pET-16b_p53). This recombinant <br /> <br /> plasmid was transformed to E. coli BL21(DE3) with and without chaperon <br /> <br /> plasmids, pGroElDnaK, and characterized using restriction analysis. The <br /> <br /> optimization of p53 overproduction was done by variation of IPTG concentration <br /> <br /> and induction temperature. High amount of p53 protein was produced at 0.7 mM <br /> <br /> IPTG concentration. Overproduction of p53 at 17°C, 25°C, and 37°C induction <br /> <br /> temperatures showed the same level of expression of p53 protein and formed a <br /> <br /> non-soluble inclusion body. The p53 protein that was coexpressed with the GroEl- <br /> <br /> DnaK chaperone protein at 25°C and 37°C indicated the presence of p53 protein <br /> <br /> in soluble form, but the majority of p53 protein existed in the inclusion body <br /> <br /> form. The p53 inclusion body was then solubilized with Tris HCl buffer pH 7.6 <br /> <br /> containing 8 M urea. The protein was renaturated by decreasing the urea <br /> <br /> concentration to 2 M. The analysis of thermal shift assay (TSA) showed different <br /> <br /> melting curves between before and after it was renatured. TSA analysis of p53 <br /> <br /> protein that interacted with HBx showed a peak with a melting point of 28°C. This <br /> <br /> p53 protein interaction information with HBx could be used to search its inhibitor <br /> <br /> candidate. <br />