Molecular Characterization of Integrated HBsAg gene and Optimation of Expression Hepatitis B surface Antigen (HBsAg) in Pichia pastoris

• Main Author: YUWITA ( NIM : 10510003) ; Pembimbing Dr. Dessy Natalia, ANITA
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/17999
• Institution: Institut Teknologi Bandung

Hepatitis B virus (HBV) causes liver imflamation which could lead to chronic hepatitis B, cirrhosis, and hepatocelullar carcinoma. WHO (2002) reported that approximately 350 million people suffer from chronic Hepatitis B in which 15–25% risk to heart disease. Vaccination is the most effective preventive efforts to reduce the number of hepatitis B infection. In Indonesia, all commercial HBV vaccines are imported. Hence, as an attempt to provide a local HBV vaccines, Hepatitis B surface Antigen (HBsAg) which is a major component in HBV vaccine, is produced in yeast Pichia pastoris. The metylotrophic yeast P. pastoris is a superior host for high level of recombinant proteins production under regulation of alcohol oxidase (AOX1) promoter. In the previous research, HBsAg gene was successfully integrated into the chromosome of P. pastoris strain KM71 by homolog recombination single cross over. The aims of this study were to determine the copy number of HBsAg gene integrated in P. pastoris chromosome by qPCR and to optimize the HBsAg expression level by methanol induction. P. pastoris KM71-26 which was capable to grow at zeocin concentration of 2000 μg/ml appeared to have 20 copy numbers of HBsAg gene based on the qPCR analysis. Optimization of HBsAg expression showed that the highest expression was achieved at 72 hours incubation with 2.5% (v/v) methanol induction. The SDS-PAGE analysis showed that the recombinant HBsAg was expressed as a monomer with molecular weight of 29 kDa and also as a dimer of 58 kDa.