通过定向突变在大肠杆菌中表达恒温 MMLV 逆转录酶。

IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Marzieh Divbandi, Ahad Yamchi, Hadi Razavi Nikoo, Abdolvahab Moradi, Alijan Tabarraei
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引用次数: 0

摘要

Moloney murine leukemia virus reverse transcriptase(MMLV RT)的功能将随着其溶解性和热稳定性的改善而增强。要实现这些特性,需要在 MMLV RT 序列的特定位置引入定向突变并优化密码子。对有定向突变(rRT-K)和无定向突变(rRT-L)的两个 RT 编码序列进行了多功能优化和表达,以分析核糖核酸酶 H(RNase H)的非活性和恒温聚合酶活性。为此,在 RT 连接域和 RNase H 活性位点分别进行了五点突变(438-442aa)和三点突变(530、568 和 659 aa)。在大肠杆菌 BL21(DE3) 和 BL21(shuffle)菌株中,37 ℃、0.5 mM IPTG 浓度和 8 小时诱导后条件下,rRT-L 和 rRT-K 获得了高表达水平。然后纯化重组酶,并通过 Ni-NTA 树脂和 Western 印迹进行验证。Insilico分析(IUpred 3.0)表明,与rRT-L相比,RNase H结构域的定向突变导致rRT-K的RNase H结构域形成紊乱区或不稳定区。改良的 RT-PCR 和 RT-LAMP 反应证明了 rRT-K 的 RNase H 缺乏活性。此外,RT-PCR 和 RT-LAMP 反应还评估了与 rRT-L 和商业 RT 相比,rRT-K 的热稳定性的提高情况。结果表明,在这两种方法中,rRT-K 都能成功耐受 60 ºC。这项研究表明,定向突变和多功能序列优化有望生产出恒温的商用酶,从而减少非特异性的一步法RT-PCR和RT-LAMP产物。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Expression of thermostable MMLV reverse transcriptase in Escherichia coli by directed mutation.

The functionality of Moloney murine leukemia virus reverse transcriptase (MMLV RT) will increase with the improvement of its solubility and thermal stability. Introduce directed mutation at specific positions of the MMLV RT sequence and codon optimization is needed to achieve these properties. The two RT coding sequences with (rRT-K) and without directed mutations (rRT-L) were versatility optimized and expressed to analyze the ribonuclease H (RNase H) inactivity and thermostable polymerase activity. For this purpose, the five-point mutations (438-442aa) and three-point mutations (530, 568, and 659 aa) were done at the RT connection domain and RNase H active site, respectively. High expression levels of rRT-L and rRT-K were obtained in E. coli BL21(DE3) and BL21(shuffle) strains, 0.5 mM IPTG concentration at 37 °C, and 8 hours' post-induction condition. Then, recombinant enzymes were purified and verified by Ni-NTA resin and western blotting. Insilico analysis (IUpred 3.0) showed that the directed mutation in the RNase H domain caused the formation of disorder regions or instability in the RNase H domain of rRT-K compared to rRT-L. The modified RT-PCR and the RT-LAMP reactions proved the RNase H inactivity of rRT-K. In addition, increasing of thermostability of rRT-K compared to rRT-L and commercial RT was evaluated by the RT-PCR and RT-LAMP reactions. The results showed that rRT-K could successfully tolerate 60 ºC in the two methods. This study revealed that the directed mutations and the versatile sequence optimization can promise to produce thermostable commercial enzymes to decrease non-specific one-step RT-PCR and RT-LAMP products.

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来源期刊
AMB Express
AMB Express BIOTECHNOLOGY & APPLIED MICROBIOLOGY-
CiteScore
7.20
自引率
2.70%
发文量
141
审稿时长
13 weeks
期刊介绍: AMB Express is a high quality journal that brings together research in the area of Applied and Industrial Microbiology with a particular interest in ''White Biotechnology'' and ''Red Biotechnology''. The emphasis is on processes employing microorganisms, eukaryotic cell cultures or enzymes for the biosynthesis, transformation and degradation of compounds. This includes fine and bulk chemicals, polymeric compounds and enzymes or other proteins. Downstream processes are also considered. Integrated processes combining biochemical and chemical processes are also published.
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