Affiliation:
1. School of Life Sciences and Technology, Bandung Institute of Technology, Bandung, Indonesia
Abstract
Introduction:
Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV
RT) is a common enzyme used to convert RNA sequences into cDNA. However, it still has
its shortcomings, especially in terms of processivity and thermostability. According to a
previous patent, the fusion of polymerase enzyme to an archaeal DNA-binding protein has
been proven to enhance its performance. Furthermore, recent studies have also stated that
the fusion of a polymerase enzyme to an archaeal DNA-binding protein is predicted to improve
its thermostability and processivity.
Aim:
As an early stage of enzyme development, this study aimed to design, express, and
purify enzymatically active MMLV RT fused with archaeal DNA-binding protein.
Methods:
RT fusion proteins were designed and evaluated using in silico methods. The RT
fusion enzyme was then expressed in Escherichia coli BL21(DE3) and purified. Its reverse
transcriptional activity was proved using reverse transcription quantitative polymerase
chain reaction (RT-qPCR).
Results:
This study showed that MMLV RT fusion with Sis7a protein at its C-terminal end
using commercial linker (GGVDMI) produced the best in silico evaluation results. The RT
fusion was successfully expressed and purified. It was also known that the optimal condition
for expression of the RT fusion was using 0.5 mM IPTG with post-induction incubation
at room temperature (± 26°C) for 16 hours. In addition, the activity assay proved that
the RT fusion has the reverse transcriptional activity.
Conclusion:
This study shows that the designed MMLV RT Sis7a fusion can be expressed
and purified, is enzymatically active, and has the potential to be developed as an improved
RT enzyme. Further study is still needed to prove its thermostability and processivity, and
further characterize, and plan production scale-up of the MMLV RT Sis7a fusion for commercial
use.
Publisher
Bentham Science Publishers Ltd.
Subject
Applied Microbiology and Biotechnology,Bioengineering,Biotechnology