Abstract：
BACKGROUND: 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.
OBJECTIVE: 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.
CONCLUSIONS: 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.
OBJECTIVE: 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.
CONCLUSIONS: 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.
摘要：
背景：莫洛尼鼠白血病病毒逆转录酶（MMLVRT）是用于将RNA序列转化为cDNA的常见酶。然而,它仍然有缺点，特别是在持续性和热稳定性方面。根据先前的专利，聚合酶与古细菌DNA结合蛋白的融合已被证明可以增强其性能。此外，最近的研究还表明，聚合酶与古细菌DNA结合蛋白的融合有望提高其热稳定性和持久性。
目的：作为酶发展的早期阶段，这项研究旨在设计，快递,并纯化与古细菌DNA结合蛋白融合的酶活性MMLVRT。
方法:使用计算机模拟方法设计和评估RT融合蛋白。然后在大肠杆菌BL21(DE3)中表达RT融合酶并纯化。使用逆转录定量聚合酶链反应（RT-qPCR）证明了其逆转录活性。
结果：这项研究表明，使用商业接头（GGVdmi）在其C末端与Sis7a蛋白的MMLVRT融合产生了最佳的计算机评估结果。成功表达并纯化了RT融合体。还已知RT融合表达的最佳条件是使用0.5mMIPTG并在室温(±26°C)下诱导后孵育16小时。此外,活性测定证明了RT融合具有反转录活性。
结论：这项研究表明，设计的MMLVRTSis7a融合体可以表达和纯化，具有酶活性，并具有作为改进的RT酶被开发的潜力。还需要进一步的研究来证明其热稳定性和持续性，并进一步表征，并计划扩大MMLVRTSis7a融合的生产规模，用于商业用途。
目的：作为酶发展的早期阶段，这项研究旨在设计，快递,并纯化与古细菌DNA结合蛋白融合的酶活性MMLVRT。
方法:使用计算机模拟方法设计和评估RT融合蛋白。然后在大肠杆菌BL21(DE3)中表达RT融合酶并纯化。使用逆转录定量聚合酶链反应（RT-qPCR）证明了其逆转录活性。
结果：这项研究表明，使用商业接头（GGVdmi）在其C末端与Sis7a蛋白的MMLVRT融合产生了最佳的计算机评估结果。成功表达并纯化了RT融合体。还已知RT融合表达的最佳条件是使用0.5mMIPTG并在室温(±26°C)下诱导后孵育16小时。此外,活性测定证明了RT融合具有反转录活性。
结论：这项研究表明，设计的MMLVRTSis7a融合体可以表达和纯化，具有酶活性，并具有作为改进的RT酶被开发的潜力。还需要进一步的研究来证明其热稳定性和持续性，并进一步表征，并计划扩大MMLVRTSis7a融合的生产规模，用于商业用途。
