OVERPRODUKSI PROTEIN X VIRUS HEPATITIS B FUSI TIOREDOKSIN PADA SUHU RENDAH DI SEL Escherichia coli BL21(DE3) DAN KARAKTERISASINYA
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...
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| Format: | Final Project |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/40460 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | 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.
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