DEVELOPMENT OF REVERSE TRANSCRIPTASE ENZYME FROM MOLONEY MURINE LEUKEMIA VIRUS FUSED WITH SIS7A PROTEIN FROM SULFOLOBUS ISLANDICUS

• Main Author: Alita Fananda, Amalia
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/74921
• Institution: Institut Teknologi Bandung

Reverse transcriptase (RT) is a polymerase enzyme that use RNA as its template. One commonly used RT enzyme is derived from the Moloney Murine Leukemia Virus (MMLV RT). MMLV RT exhibits higher catalytic activity and fidelity processivity compared to other RT enzymes. However, its low processivity and thermolability necessitates further development to enhance its characteristics for better performance. Previous research has designed a fusion between the MMLV RT enzyme and the 7 kDa DNA-binding protein from Archaea Sulfolobus islandicus, known as Sis7a. In silico analysis of the fusion enzyme indicated improved structural stability and ligand affinity compared to wild-type MMLV RT. Additionally, expression and purification optimization were performed to obtain the soluble fraction of the fusion enzyme. However, further characterization of the recombinant RT enzyme is required. In this study, we aimed to purify and characterize the MMLV RT enzyme fused with the Sis7a protein from Archaea Sulfolobus islandicus. The recombinant protein, expressed in Escherichia coli BL21 (DE3), was purified using chromatography columns and confirmed through western blotting. Several enzyme parameters were assessed, including relative unit activity, thermostability, the optimum reaction temperature, processivity, and inhibitor resistance. The commercial MMLV RT enzyme (RTK) was used as a positive control. Production of the fusion RT enzyme (RTF) in the soluble condition was successfully achieved, confirmed by the presence of the target band in western blot analysis using His-tag antibodies. The enzyme's activity was evaluated by comparing Cq values with the no template control (NTC) and RTK, and the presence of amplified cDNA bands was observed through agarose gel electrophoresis. Notably, a production yield of approximately 23116 U/?l RTF was obtained. The PCR efficiency when using RTF was calculated as 91.53%, with an R2 value of 0.9835. The optimum reaction temperature of RTF was determined to be around 45°C, within a range of 40°C to 50°C. The thermostability test determined melting temperature (Tm) of approximately 50°C for RTF. RTF exhibited resistance to various inhibitors, including guanidine isothiocyanate, whole blood, blood plasma, ethanol, and urea. Meanwhile, processivity value was not obtained in this study. In conclusion, this study successfully characterized the fusion RT enzyme and its parameter values comparable to commercial RT enzyme. Further research should focus on scaling up the production of this RTF protein for potential application in diagnostic kits.