EKSPRESI PROTEIN CYP7A1, ENZIM KUNCI DALAM BIOSINTESIS SENYAWA ASAM URSODEOKSIKOLAT

• Main Author: Rabila Firly, Nayla
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/43986
• Institution: Institut Teknologi Bandung

Cholestatic liver disease is a condition where bile flow disorders occur. The disorder can occur due to lack of bile or there is a blockage in the bile duct. One treatment that can alleviate this disease is by consuming ursodeoxycholic acid or ursodeoxycholic acid (UDCA). In general, UDCA can be found in humans but in limited quantities. The 7-?-hydroxylase cholesterol enzyme is an enzyme that plays an important role in the UDCA biosynthesis of cholesterol and is encoded by the cytochrome P450 7A1 gene (CYP7A1). In previous studies, the CYP7A1 gene was cloned from Gallus gallus chicken liver into Escherichia coli DH5?. In addition, previous studies have also carried out the construction of the CYP7A1 gene into pET-32b(+) expression vectors but there were few construction errors. The purpose of this study is to know CYP7A1 gene expression after construction by the site-directed mutagenesis. In this study, a positive plasmid inserted by the CYP7A1 gene will be used as a DNA template for mutation stages. The mutation is done by adding one base in the form of base G before the start codon of the CYP7A1 gene. Therefore, used primers that have been designed for mutation stages. Recombinant plasmid pET-CYP7A1 which has been mutated will be transformed into Escherichia coli DH5?with the selection of ampicillin antibiotics. Confirmation of the success of the transformation was carried out by the PCR method where the results of the method showed the presence of the CYP7A1 gene. The plasmid pET-CYP7A1 mutation will be transformed into E. coli BL21(DE3) with the selection of ampicillin antibiotics for expression purposes. Positive colonies containing pET-CYP7A1 will be induced by 1 mM IPTG to see the expression given and compared with controls and non-mutation samples induced by 1 mM IPTG and not induced by 1mM IPTG. Analysis of CYP7A1 gene expression was performed by the SDS- PAGE method where there was a band at a size of about 70 kDa.