Improvement of Moloney murine leukemia virus reverse transcriptase thermostability by introducing a disulfide bridge in the ribonuclease H region

被引：7

作者：

Narukawa, Yutaro ^{[1
]}

Kandabashi, Mako ^{[1
]}

Li, Tongyang ^{[1
]}

Baba, Misato ^{[1
]}

Hara, Haruka ^{[1
]}

Kojima, Kenji ^{[1
]}

Iida, Kei ^{[2
]}

Hiyama, Takayoshi ^{[3
]}

Yokoe, Sho ^{[3
]}

Yamazaki, Tomomi ^{[3
]}

Takita, Teisuke ^{[1
]}

Yasukawa, Kiyoshi ^{[1
]}

机构：

[1] Kyoto Univ, Grad Sch Agr, Div Food Sci & Biotechnol, Sakyo Ku, Kyoto 6068502, Japan

[2] Kyoto Univ, Med Res Support Ctr, Grad Sch Med, Sakyo Ku, Yoshida Konoe Cho, Kyoto 6068501, Japan

[3] Toyobo Co Ltd, Tsuruga Inst Biotechnol, 10-24 Toyo Cho, Tsuruga, Fukui 9148550, Japan

来源：

PROTEIN ENGINEERING DESIGN & SELECTION | 2021年 / 34卷

基金：

日本学术振兴会;

关键词：

disulfide bridge; Moloney murine leukemia virus; site-directed mutagenesis; reverse transcriptase; thermostability; THERMAL-STABILITY; CRYSTAL-STRUCTURE; GENERATION; INCREASE; MUTAGENESIS; MUTATIONS; XYLANASE;

D O I：

10.1093/protein/gzab006

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页数：8

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