In:
Recent Patents on Biotechnology, Bentham Science Publishers Ltd., Vol. 17 ( 2023-04-03)
Abstract:
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. Meanwhile, the fusion of a polymerase enzyme to an archaeal DNA-binding protein is predicted to improve
its thermostability and processivity. background: Moloney murine leukemia virus reverse transcriptase (MMLV RT) is an enzyme used to convert RNA sequences into cDNA, commonly used in industrial settings and for engineering purposes. However, it still has its own shortcomings, especially in terms of processivity and thermostability. Meanwhile, fusion of a polymerase enzyme to an archaeal DNA-binding protein is predicted to improve its thermostability and processivity. Introduction: 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. objective: Since the enzyme was still in its early stages of development, this study''s objective is to do in silico design evaluation, determine the optimal conditions for the RT fusion expression and purification, and to also prove its reverse transcriptional activity using reverse transcription quantitative polymerase chain reaction (RT-qPCR). 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). method: Enzyme design was done using in silico methods. Protein expression was done by variations in post-induction conditons, purification was done using affinity column, and reverse transcriptional activity assay was done 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. other: Further study is still needed to prove the thermostability and processivity, further characterize, and plan production scale up of the MMLV RT-Sis7a fusion for commercial use.
Type of Medium:
Online Resource
ISSN:
1872-2083
DOI:
10.2174/1872208317666230403104302
Language:
English
Publisher:
Bentham Science Publishers Ltd.
Publication Date:
2023
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