通过在核糖核酸酶 H 区域引入二硫键来提高 Moloney 鼠白血病病毒逆转录酶的热稳定性。
Improvement of Moloney murine leukemia virus reverse transcriptase thermostability by introducing a disulfide bridge in the ribonuclease H region.
机构信息
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
Medical Research Support Center, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
出版信息
Protein Eng Des Sel. 2021 Feb 15;34. doi: 10.1093/protein/gzab006.
Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) is widely used in research and clinical diagnosis. Improvement of MMLV RT thermostability has been an important topic of research for increasing the efficiency of cDNA synthesis. In this study, we attempted to increase MMLV RT thermostability by introducing a disulfide bridge in its RNase H region using site-directed mutagenesis. Five variants were designed, focusing on the distance between the two residues to be mutated into cysteine. The variants were expressed in Escherichia coli and purified. A551C/T662C was determined to be the most thermostable variant.
莫洛尼鼠白血病病毒（MMLV）逆转录酶（RT）被广泛应用于科研和临床诊断。提高 MMLV RT 的热稳定性一直是提高 cDNA 合成效率的一个重要研究课题。本研究通过定点突变技术在其 RNase H 区引入二硫键，试图提高 MMLV RT 的热稳定性。设计了 5 种变体，重点关注要突变为半胱氨酸的两个残基之间的距离。这些变体在大肠杆菌中表达并纯化。A551C/T662C 被确定为最耐热的变体。
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