THE OPTIMIZATION OF INCUBATION TEMPERATURE TO EXPRESS HIV PROTEASE GEN IN Escherichia coli BL21 (DE3) FOR DEVELOPING ANTI HIV DRUG SELECTION SYSTEM

• Main Author: Wirdullutfi
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/41224
• Institution: Institut Teknologi Bandung

People with HIV / AIDS in Indonesia in 2017 amounted to 630,000 people with 49,000 new patients with 39,000 deaths. Of the number of HIV sufferers in Indonesia, patients who received antiretroviral therapy (ART) were only as much as 14.5% (91,400 people). One problem for reducing the number of people with HIV is expensive medical expenses, so the search for drugs that can further reduce medical costs is an important priority including the search for drugs from natural ingredients. Indonesia is a country with high biodiversity so that it can be an alternative source of medicine. A method that to select anti-HIV drugs is the cross-linking dimerization. However, this method is not get applicable due to the unavailability of HIV recombinant protease which is the main component in the method. Therefore, in this study, we optimized the expression of the HIV protease gene in E.coli recombinant bacteria. In the previous study, the HIV protease coding gene had been successfully inserted in the pTXB1 vector and had confirmed the presence of the gene insert. Hence in this study temperature optimization was carried out to determine the best temperature to be used in expressing HIV protease proteins. In this study, incubation was carried out in two treatment groups, namely, the group with the addition of 1mM IPTG (induction) and the group without the addition of IPTG (non-induction). In the non-induction and induction groups, recombinant bacterial cultures were incubated for 16 hours, then subcultured. After OD reaches 0.4, the culture is divided into three groups of incubation temperatures of 20, 25 and 37oC. In cultures not induced by IPTG, incubation was carried out at a temperature of 20oC for 12 hours, at a temperature of 25oC for 6 hours, and at 37oC for 3 hours. In bacterial cultures given IPTG induction, incubation was carried out at 20oC for 8 hours, at 25oC for 4 hours, and at 37oC for 2 hours. After reaching their respective incubation times, the total protein from each group was isolated and a sonication method was performed to separate the protein in the dissolved phase and the non-dissolved phase. Then analyzed using the method of Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The visualization results in each treatment group, both in the dissolved and non-dissolved phases showed protein expression at temperatures of 20oC, 25oC, and 37oC with the formation of protein bands of the expected size of 35.3kDa. Therefore, in this study, it can be concluded that the induction of IPTG, temperature and incubation time did not to affect the expression of recombinant HIV proteases in E. coli bacteria.