OVERPRODUCTION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT AVIAN MYELOBLASTOSIS VIRUS REVERSE TRANSCRIPTASE ?- SUBUNIT IN ESCHERICHIA COLI BL21-CODONPLUS (DE3)-RIPL
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....
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| Format: | Theses |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/54416 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | 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.
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