OVERPRODUCTION, PURIFICATION, AND CHARACTERIZATION X PROTEIN HEPATITIS B VIRUS FUSED THIOREDOXIN IN ESCHERICHIA COLI ROSETTA-GAMI

• Main Author: Hadianti, Tia
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/48682
• Institution: Institut Teknologi Bandung

Hepatitis B virus (HBV) cause 96% of hepatitis mortality worldwide. HBV infection leads to chronic hepatitis progression into cirrhosis and liver carcinoma. HBV X protein (HBx) is known to contribute to the pathogenesis of liver carcinoma. HBx structure consist 4 disulfide bonds and 53.7% hydrophobic amino acids causes HBx expression in Escherichia coli as inclusion bodies (IB). The aim of this study to overproduce, solubilize, renaturate, and purify HBx fused Thioredoxin (Trx-HBx). In this study, Trx-HBx was expressed in E. coli Rosettagami at low temperatures. Overproduction of protein was optimized in various induction temperature (17°C and 25°C) and various induction time (8 and 16 hours). The protein was characterized by SDS-PAGE, dot blot, and Western blot. The results of this study showed that the majority of Trx-HBx was expressed as IB under optimum temperature at 25°C and 16 hours induction. Trx-HBx was solubilized from IB using 6 M urea solubilization buffer. Solubilized Trx-HBx was renatured simultaneous with purification using affinity chromatography nickel column. Trx-HBx was purified using 200 mM imidazole at the fourth and fifth elution, but SDS-PAGE reducing and non-reducing did not show disulfide bond formation after renaturation.