Improvement of Moloney murine leukemia virus reverse transcriptase thermostability by introducing a disulfide bridge in the ribonuclease H region-Reference-Cited by-同舟云学术

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

Published:2021 Issue: Volume:34 Page:
ISSN:1741-0126
Container-title:Protein Engineering, Design and Selection
language:en
Short-container-title:

Author:

Narukawa Yutaro¹,Kandabashi Mako¹,Li Tongyang¹,Baba Misato¹,Hara Haruka¹,Kojima Kenji¹,Iida Kei²,Hiyama Takayoshi³,Yokoe Sho³,Yamazaki Tomomi³,Takita Teisuke¹,Yasukawa Kiyoshi¹^ORCID

Affiliation:

1. Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan

2. Medical Research Support Center, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan

3. Tsuruga Institute of Biotechnology, Toyobo Co., Ltd. 10-24 Toyo-cho, Tsuruga 914-8550, Japan

Abstract

Abstract 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.

Funder

Grants-in-Aid for Scientific Research

Japan Society for the Promotion of Science

Publisher

Oxford University Press (OUP)

Subject

Molecular Biology,Biochemistry,Bioengineering,Biotechnology

Link

http://academic.oup.com/peds/article-pdf/doi/10.1093/protein/gzab006/36955028/gzab006.pdf

Reference30 articles.

1. Novel mutations in Moloney Murine Leukemia Virus reverse transcriptase increase thermostability through tighter binding to template-primer

2. Further increase in thermostability of Moloney murine leukemia virus reverse transcriptase by mutational combination