The attachment of a DNA-binding Sso7d-like protein improves processivity and resistance to inhibitors of M-MuLV reverse transcriptase

Igor P. Oscorbin, Pei Fong Wong, Ulyana A. Boyarskikh, Evgeny A. Khrapov, Maksim L. Filipenko

Research output: Contribution to journalArticlepeer-review

Abstract

Reverse transcriptases (RTs) are a standard tool in both fundamental studies and diagnostics. RTs should possess elevated temperature optimum, high thermal stability, processivity and tolerance to contaminants. Here, we constructed a set of chimeric RTs, based on the combination of the Moloney murine leukaemia virus (M-MuLV) RT and either of two DNA-binding domains: the DNA-binding domain of the DNA ligase from Pyrococcus abyssi or the DNA-binding Sto7d protein from Sulfolobus tokodaii. The processivity and efficiency of cDNA synthesis of the chimeric RT with Sto7d at the C-end are increased several fold. The attachment of Sto7d enhances the tolerance of M-MuLV RT to the most common amplification inhibitors: NaCl, urea, guanidinium chloride, formamide, components of human whole blood and human blood plasma. Thus, fusing M-MuLV RT with an additional domain results in more robust and efficient RTs.

Original languageEnglish
Pages (from-to)4338-4356
Number of pages19
JournalFEBS Letters
Volume594
Issue number24
Early online date24 Sep 2020
DOIs
Publication statusPublished - Dec 2020

Keywords

  • cDNA synthesis
  • chimeric protein
  • M-MuLV reverse transcriptase
  • protein engineering
  • reverse transcription
  • VIRUS
  • RNA
  • PERFORMANCE
  • POLYMERASE
  • AVIAN-MYELOBLASTOSIS
  • AMPLIFICATION
  • INCREASE
  • GENERATION
  • THERMOSTABILITY
  • RIBONUCLEASE

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