OPTIMIZATION CONCENTRATION AND INCUBATION TIME OF IPTG IN EXPRESSION OF HIV-1 PROTEASE BY Escherichia coli BL21 (DE3) FOR DEVELOPMENT OF ANTI-HIV DRUG SCREENING METHODS

• Main Author: Nur'Azmi, Akifah
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/39567
• Institution: Institut Teknologi Bandung

Human Immunodeficiency Virus (HIV) is a virus that attacks the immune system. It is known that infection with the virus has a high mortality rate. Although HIV infection cannot be completely removed from the patient's body, however, at this time the infection has been controlled using antiretroviral therapy (ART). This treatment is used to control HIV replication in patients. One group of ART drugs that has a high genetic barrier is protease inhibitors. The target of this treatment is HIV-1 protease which is an enzyme that plays an important role in the maturation of the HIV virus. However, the availability of ART is still very limited, especially in developing countries such as Indonesia. So it is necessary to develop new anti- HIV drugs to answer those needs. One method of developing anti-HIV drugs is screening of candidate drug compounds that can deactivate HIV proteases by preventing the dimerization of these proteins. However, in the method of screening these drugs, a large number of HIV proteases are needed. Therefore, this study will optimize the production of HIV-1 proteases using the expression system Escherichia coli BL21 (DE3) which will be used in the development of screening methods for anti-HIV drugs. The HIV-1 protease gene was constructed on pTXB1 plasmid and subsequently transformed into Escherichia coli BL21 (DE3). The success of this transformation was confirmed using polymerase chain reaction (PCR) and sequencing methods. Recombinant protein is then expressed from transformant culture. Protein expression was carried out at an incubation temperature of 37ºC and optimized by varying the IPTG concentrations at 0, 1, 2, 3, 4, and 5 mM and the incubation time of IPTG at 0,1,2,3 and 4 hours. Protein analysis was carried out by SDS PAGE method and the measurement of protein band intensity was quantified using Image J software. Based on the research, plasmid confirmation results showed that the presence of a protease gene measuring around 300 bp. From the results of protein analysis, HIV-1 proteases have a size of about 35 kDa and can be expressed without the addition of IPTG inductors. The optimum IPTG concentration to express HIV protease in Escherichia coli BL21 (DE3) in the supernatantt fraction was 1 mM and the optimum concentration of IPTG in the pellet fraction could not be determined, with optimum incubation time, in pellet or supernatantt fractions, 1 hour. For cells that were not induced by IPTG, optimum protein expression occurred at the 3rd hour for the pellet fraction and at the 4th hour for the supernatantt fraction. This study shows that HIV-1 proteases in the plasmid pTXB-HIV construct can be expressed in the E. coli BL21 (DE3) system. In addition, several alternative conditions for optimum culture for expressing recombinant HIV-1 proteases have also been obtained. Future studies are needed to see the inhibitory activity of dimerization of the recombinant protease.