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. 2020 Dec;594(24):4338-4356.
doi: 10.1002/1873-3468.13934. Epub 2020 Oct 5.

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

Igor P Oscorbin  1   2 , Pei Fong Wong  1   2 , Ulyana A Boyarskikh  1   2 , Evgeny A Khrapov  1 , Maksim L Filipenko  1   2
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Free article

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

Igor P Oscorbin et al. FEBS Lett. 2020 Dec.
Free article

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.

Keywords: M-MuLV reverse transcriptase; cDNA synthesis; chimeric protein; protein engineering; reverse transcription.

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