EXPRESSION, PURIFICATION, AND ENZYME ACTIVITY ASSAY OF MOLONEY MURINE LEUKEMIA VIRUS FUSED WITH SIS7A PROTEIN FROM SULFOLOBUS ISLANDICUS

• Main Author: Marthasya Simanjuntak, Goldyna
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/66841
• Institution: Institut Teknologi Bandung

Reverse transcriptase (RT) is an enzyme that converts RNA sequences into cDNA. However, this RT enzyme is known to still have shortcomings, especially in processivity and thermostability. Therefore, previous in silico study was conducted to design a recombinant RT that would increase both drawbacks. The recombinant RT enzyme designed in the previous study was Moloney Murine Leukemia Virus (MMLV) RT fused with Sis7a protein from Sulfolobus islandicus at the C-terminal end. However, the designed MMLV RT fusion has not yet been tested in vitro. Therefore, in this study, the conditions for its expression were optimized and the enzyme activity assay of the RT fusion enzyme was carried out in vitro. The designed MMLV RT fusion enzyme was constructed on the pET-23a(+) plasmid. The constructed plasmid was cloned in Escherichia coli DH5? and then expressed in E. coli BL21(DE3). The transformation was carried out using the heat-shock method at 42°C with variations in duration (55, 60, 75, and 90 seconds) and in the plasmid concentrations (4, 100, 200, and 800 ng). The transformation process was confirmed using Polymerase Chain Reaction (PCR) and DNA sequencing. Expression of recombinant protein was carried out at room temperature with variations in isopropyl ?-d-1-thiogalactopyranoside (IPTG) concentrations (0,25, 0,5, and 0,75 mM) as well as in the durations of induction (4, 5, and 16 hours). Protein expression analysis was performed using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Purification of recombinant protein was carried out with a Ni-NTA column. Then enzyme activity assay was carried out using two different buffers: phosphate-buffered saline (PBS) and detergent-based. The purified recombinant enzymes were then concentrated prior to the enzyme activity assay. The recombinant enzyme activity assay was carried out using the reverse transcription-quantitative PCR (RT-qPCR) method which targets RNA from the SARS-CoV-2. This study shows that the transformation to E. coli BL21(DE3) was successfully carried out at the temperature of 42°C for 90 seconds with 800 ng plasmid concentration, as proved with PCR and DNA sequencing. The optimum expression conditions were obtained with incubation at room temperature with a duration of 16 hours and the addition of 0.5 mM IPTG, even though the addition of IPTG under these conditions did not significantly increase the expression of recombinant protein. The results of the enzyme activity assay using RT-qPCR showed that the recombinant enzyme had reverse transcription activity with the best activity obtained using PBS buffer. So it can be concluded that the MMLV RT enzyme fusion with Sis7a protein from Sulfolobus islandicus was expressed in E. coli BL21(DE3) and was proved to have reverse transcriptional activity. Further research is needed to prove processivity, thermostability, determine the optimum reaction’s condition, and plan optimization research for scaling up.