OVERPRODUCTION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT MOLONEY MURINE LEUKEMIA VIRUS REVERSE TRANSCRIPTASE

• Main Author: Fajrul Ihsan, Ardika
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/56619
• Institution: Institut Teknologi Bandung

Reverse transcriptase (RTase) is a protein that catalyzes the synthesis of DNA from the RNA template, and is an important component of Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR). RTase from Moloney Murine Leukemia Virus (MMLV) mutant (RTM) is resistant to the RT-qPCR process inhibitor and does not have RNase H activity so that it can increase cDNA yield from the reverse transcription process. This study aims to overproduce, purify and characterize recombinant RTM protein in Escherichia coli BL21(DE3) in order to obtain functional RTM protein that can be used in the process of in vitro reverse transcription. RTM protein was constructed using codon optimization approach using plasmid pET-28a with the Codon Adaptation Index (CAI) of 0.773 and the %GC of 54.7%. Overproduction of RTM was carried out by co-expression of Trigger Factor (TF) and GroEL-GroES. Overproduction of RTM protein at optimal temperature parameters of 28 ?, 0.5 mM of IPTG and 20 ng/mL of tetracycline induction showed a RTM soluble fraction of 94.2 ± 1.73% and a yield of 39.22 ± 4.63%. Purification of RTM was then carried out using Ni-NTA Sepharose affinity column chromatography with 250 mM imidazole elution. Pure RTM was obtained with a yield of 42.58 ± 3.27% and a purity level of 88.73 ± 3.19%. Activity determination and the tests of storage stability and influence of the presence of RTase inhibitors and clinical samples were carried out using the RT-qPCR and SDS-PAGE methods. RTM protein has RNA-dependent DNA polymerase activity with a specific activity of 1981,03 U/mg, and is stable for 15 weeks at -20 ? storage condition. In addition, RTM remained active in the presence of RT-qPCR inhibitors, such as VTM SARS-CoV-2 up to 39%, and clinical samples in the form of blood plasma up to 5%, and whole blood up to 2%. The process of overproduction, purification, and characterization of RTM protein has been successfully carried out with the stability and good resistance to RTase inhibitor characteristics of the protein showing that the protein can be used in the process of in vitro reverse transcription.