OVERPRODUCTION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT AVIAN MYELOBLASTOSIS VIRUS REVERSE TRANSCRIPTASE ?- SUBUNIT IN ESCHERICHIA COLI BL21-CODONPLUS (DE3)-RIPL

• Main Author: Muazarah, Sellma
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/54416
• Institution: Institut Teknologi Bandung

Reverse transcriptase from Avian myeloblastosis virus (AMV RTase) enzyme is used to synthesize cDNA from RNA. RTase enzyme is mostly used in reverse transcription real-time PCR (RT-qPCR). RT-qPCR is widely used for detection of disease caused by RNA viruses and quantification of gene expression. The aim of this study was to overproduce recombinant AMV RTase enzyme, purifiy recombinant AMV RTase enzyme, and characterize it using activity study. Overproduction of AMV RTase enzyme was performed using Escherichia coli BL21-Codonplus (DE3)-RIPL as the host. Optimation of overproduction conditions were performed at various IPTG concentrations (0,5 and 1 mM IPTG), induction temperature (temperature 30 °C and 37 °C), and induction time (4 and 16 hours). The AMV RTase enzyme was characterized using SDS-PAGE analysis and RTqPCR. The results of this study showed that overproduction of the AMV RTase enzyme in E. coli BL21-Codonplus (DE3)-RIPL was optimum at 1 mM induction of IPTG, temperature 37°C for 4 hours. Most of the AMV RTase produced using this condition was insoluble and present in inclusion body. The inclusion bodies containing AMV RTase was successfully solubilized in 6 M urea solubilization buffer. Solubilized AMV RTase was purified simultaneously with renaturation using nickel column chromatography. The AMV RTase enzyme was successfully purified in 1 M imidazole. The activity of purified AMV RTase enzyme was performed using RT-qPCR. The RT-qPCR results showed that there was amplification signal using the RNA N-nCoV from in vitro transcription as template. AMV RTase has activity. As conclusion, the AMV RTase produced in this study showed activity, although the activity was not yet equal to the commercially available kit.