OVERPRODUKSI PROTEIN X VIRUS HEPATITIS B FUSI TIOREDOKSIN PADA SUHU RENDAH DI SEL Escherichia coli BL21(DE3) DAN KARAKTERISASINYA

• Main Author: Nadhira Aliya Putri, Aghnia
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/40460
• Institution: Institut Teknologi Bandung

Hepatitis B infects more than two billion people and causes deaths of up to one million people per year. Hepatitis B Virus (HBV) infection’s is a major risk factor in the occurrence of liver carcinomas. HBV can cause liver carcinomas through various mechanisms, including through protein X VHB (HBx). Overproduction of HBx proteins are performed using Escherichia coli BL21 (DE3) because it is widely used and produce more recombinant proteins. HBx protein expression in E. coli causes proteins to form an inclusion body. HBx protein expression in soluble and active form could improve research related to its interaction with p53 and for the discovery of inhibitor candidates. The objective of this research was to improve the soluble expression of HBx proteins in E. coli using Thioredoxin fusion protein and overproduction at low temperatures, as well as its characterization. Optimization of the Trx-HBx overproduction was carried out on two parameters, which was IPTG concentration and overproduction temperature. Trx-HBx protein was optimally induced using 0.1 mm IPTG. The optimization of overproduction temperature was carried out at 17°C and 25°C and the result showed that the Trx-HBx protein was produced mostly as inclusion body. Overproduction of Trx-HBx at 25°C was produced at 71.37% as inclusion body and 28.63% in the supernatant. The inclusion body was then washed using Triton X-100 1% and Tris HCl pH 7.4, followed by solubilization using 6 M urea. Protein was then renatured by buffer exchange to reduce the concentration of urea and then characterized using reducing and nonreducing SDS-PAGE. The results showed a different migration pattern of the Trx-HBx in reducing and nonreducing conditions, indicating the presence of disulfide bonds.