ENHANCEMENT OF SOLUBLE HBcAg PROTEIN EXPRESSION THROUGH THE USE OF LAMBDA PROMOTER

• Main Author: NATHANAEL IMAN (10413008 ), MARVIN
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/22992
• Institution: Institut Teknologi Bandung

Hepatitis B is one of the most common and deadliest liver diseases in the world. This liver disease, which is caused by the infection of Hepatitis B Virus (HBV), can lead to liver carcinoma and cirrhosis. Several alternatives are available to eliminate hepatitis B virus from the body of a patient, one of which is through the use of Hepatitis B core antigen (HBcAg)-based therapeutic vaccine, which induces the proliferation of cytotoxic T cells. Production of such vaccine requires HBcAg protein in its soluble form, expressed by vectors such as Escherichia coli. Previous studies showed low levels of HBcAg solubility expressed through T7 promoter in a bacterial vector. One approach to increase the production of soluble HBcAg protein is through the substitution of the promoter into one with a lower strength level. Weaker promoter lowers the rate of transcription and polypeptide synthesis which enable chaperone proteins to better fold polypeptides, which then results in higher levels of protein solubility. In this research, enhancement of soluble HBcAg expression is attempted through the use of lambda promoter, a relatively weak, heat-shock inducible promoter. The DNA sequence of lambda promoter is synthesized and inserted into a pET-28a (+) expression vector containing HBcAg coding sequence, replacing the original T7 promoter. The plasmid is then transformed into Escherichia coli BL21(DE3) for the HBcAg to be expressed. The protein expression is then induced through a heat-shock from room temperature into 42°C. The expressed proteins are then separated into soluble and insoluble fractions to then be analyzed using SDS-PAGE analyses. The percentage of soluble HBcAg to total protein is calculated using ImageJ software. SDS-PAGE analysis showed 21 kDa protein bands, in line with the theoritical molecular weight of HBcAg protein. Further in silico analyses showed HBcAg solubility expressed through lambda promoter reaches 49,2% while HBcAg expressed through T7 promoter only reaches 11,7%. Other result showed soluble HBcAg expressed through lambda promoter accounts for 9,8% of the total soluble protein while HBcAg expressed through T7 promoter only accounts for 5,5%. In conclusion, expression of soluble HBcAg is succesfully enhanced through the use of lambda promoter.