热稳定性
融合蛋白
逆转录酶
生物
分子生物学
过程性
小鼠白血病病毒
互补DNA
聚合酶
DNA聚合酶
DNA
酶
生物化学
聚合酶链反应
基因
重组DNA
作者
Goldyna M Simanjuntak,Azzania Fibriani,Amalia A Fananda,Nicholas Yamahoki
出处
期刊:Recent Patents on Biotechnology
[Bentham Science]
日期:2024-03-01
卷期号:18 (1): 71-83
标识
DOI:10.2174/1872208317666230403104302
摘要
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.
科研通智能强力驱动
Strongly Powered by AbleSci AI