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Biotechnology Letters

, Volume 34, Issue 7, pp 1209–1215 | Cite as

Improving the thermal stability of avian myeloblastosis virus reverse transcriptase α-subunit by site-directed mutagenesis

  • Atsushi Konishi
  • Kiyoshi YasukawaEmail author
  • Kuniyo Inouye
Original Research Paper

Abstract

Avian myeloblastosis virus reverse transcriptase (AMV RT) is a heterodimer consisting of a 63 kDa α-subunit and a 95 kDa β subunit. Moloney murine leukaemia virus reverse transcriptase (MMLV RT) is a 75 kDa monomer. These two RTs are the most extensively used for conversion of RNA to DNA. We previously developed several mutations that increase the thermostability of MMLV RT and generated a highly stable MMLV RT variant E286R/E302K/L435R/D524A by combining three of them (Glu286→Arg, Glu302→Lys, and Leu435→Arg) and the mutation to abolish RNase H activity (Asp524→Ala) [Yasukawa et al. (2010) J Biotechnol 150:299–306]. To generate a highly stable AMV RT variant, we have introduced the triple mutation of Val238→Arg, Leu388→Arg, and Asp450→Ala into AMV RT α-subunit and the resulted variant V238R/L388R/D450A, was expressed in insect cells and purified. The temperature decreasing the initial activity by 50 %, measured over 10 min, of the variant with or without template primer (T/P), poly(rA)-p(dT)15, was 50 °C; for the wild-type AMV RT α-subunit (WT) this was 44 °C. The highest temperature at which the variant exhibited cDNA synthesis activity was 64 °C; the WT was 60 °C. A highly stable AMV RT α-subunit is therefore generated by the same mutation strategy as applied to MMLV RT and that positive charges are introduced into RT at positions that have been implicated to interact with T/P by site-directed mutagenesis.

Keywords

cDNA synthesis Avian myeloblastosis virus Reverse transcriptase Site-directed mutagenesis Stability 

Abbreviations

AMV

Avian myeloblastosis virus

MMLV

Moloney murine leukaemia virus

RT

Reverse transcriptase

T/P

Template-primer

Notes

Acknowledgments

This study was supported in part (K.Y.) by Grants-in-Aid for Scientific Research (nos. 19580104 and 21580110) from the Japan Society for the Promotion of Science and Daiwa Securities Health Foundation.

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Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Atsushi Konishi
    • 1
  • Kiyoshi Yasukawa
    • 1
    Email author
  • Kuniyo Inouye
    • 1
  1. 1.Division of Food Science and Biotechnology, Graduate School of AgricultureKyoto UniversitySakyo-kuJapan

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