PRODUCTION OF RECOMBINANT E2 PROTEIN FROM CHIKUNGUNYA VIRUS (CHIKV) IN Escherichia coli

• Main Author: Muarif, Agam
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/38309
• Institution: Institut Teknologi Bandung

“Chikungunya” is adapted from Swahili (Tanzania) which means bending or curving upward. Chikungunya was discovered in 1952 in southern Tanzania and spread to Africa, Asia, America, Europe, and Oceania/Pacific islands. Indonesia is one of the Asian countries infected by chikungunya. Chikungunya is a disease caused by chikungunya virus (CHIKV), that shown with symptoms such as fever, pain and swelling in the joints, also rash on the skin. CHIKV is included in the genus Alphavirus and family Togaviridae. The spread of CHIKV occurs through the bite of Aedes aegypti and Aedes albopictus mosquitoes. In Indonesia, CHIKV has been spread since 1973 until now. CHIKV genome size is 11.8 kilobase and consists of two open reading frames (ORF) encoding four non-structural proteins (nsP1, nsP2, nsP3, nsP4) and five structural proteins (capsid, E3, 6K, E1, E2). On the life cycle alphavirus the structural E2 proteins had a role in the binding of the host cell receptors. The E2 envelope protein has immunogenicity towards the host cell and induces the producing of antibodies. A high immunogenicity level and can detection antibody IgM anti-CHIKV of E2 envelope protein can be used as a component for dignostic kit and is an important component in making vaccines chikungunya. This study aims to express gene encoding recombinant E2 protein from CHIKV in Escherichia coli. E. coli BL21 (DE3) was used as a host cell to make CHIKV E2 protein because it had the ?DE3 gene encoding the T7 RNA polymerase that could express the gene targets in expression vectors by inducer isopropyl-?-D-thiogalactoside (IPTG). In this study, gene encoding the CHIKV E2 protein is chemically synthesized (synthetic gene) before being inserted into the expression vector pET-16b. The E. coli BL21 (DE3) transformation was performed with a plasmid pET16b-E2 resulting in colonies containing pET16b- E2. The expression analysis of two recombinant colonies (colonies 3 and 5) was performed by optimizing induction temperatures (4°, 18° and 37°C) and optimizing the concentration of IPTG (0.1 and 0.5 mM). Incubation time after induction IPTG at 4°C is 24 hours, at 37°C is 4 hours and at 18°C uses seven variables of incubation time, that’s are 1, 2, 3, 4, 5, 6, and 24 hours. This analysis is mandatory for the maximum conditions to be used on the gene encoding the E2 protein expression. The results of this analysis all of conditions optimization gene encoding of the E2 protein expression were characterized using Sodium Dodecyl Sulfate Polyacrilamide Gel Electrophoresis (SDS PAGE). The optimum condition of E2 recombinant protein expression obtained was at colony 5, 37°C and 0.1 mM IPTG concentration. This optimum conditions is seen quantitatively based on the thick of the band on SDS-PAGE electrophoregram with a molecular weight of ~47 kDa corresponding to the E2 protein. The results of the expression of the recombinant gene of E2 CHIKV are the insoluble protein in buffer (inclusion bodies). This result is seen on SDS-PAGE electrophoregram from cell pellet of the cell lysis and not on the supernatant of the cell lysis. Protein expression result then refolded by the dilution flash method to get the E2 protein soluble in the buffer. This method is done by reducing the concentration of denaturant (urea) so that the protein obtained in the folded state. The refolding protein result is soluble in the buffer with concentration 22,2 ?g/mL and sized ~ 47 kDa on SDS-PAGE electrophoregram.