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

Protein Eng Des Sel. 2021 Feb 15:34:gzab006. doi: 10.1093/protein/gzab006.

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.

Keywords: Moloney murine leukemia virus; disulfide bridge; reverse transcriptase; site-directed mutagenesis; thermostability.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Disulfides
  • Mice
  • Moloney murine leukemia virus* / genetics
  • Mutagenesis, Site-Directed
  • RNA-Directed DNA Polymerase* / genetics
  • Ribonuclease H / genetics

Substances

  • Disulfides
  • RNA-Directed DNA Polymerase
  • Ribonuclease H