Improving the thermal stability of avian myeloblastosis virus reverse transcriptase α-subunit by site-directed mutagenesis
- 476 Downloads
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.
KeywordscDNA synthesis Avian myeloblastosis virus Reverse transcriptase Site-directed mutagenesis Stability
Avian myeloblastosis virus
Moloney murine leukaemia virus
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.
- Gerard GF (1998) Reverse transcriptase: a historical perspective. Focus 20:65–67Google Scholar
- Ishiguro T, Saitoh J, Horie R, Hayashi T, Ishizuka T, Tsuchiya S, Yasukawa K, Kido T, Nakaguchi Y, Nishibuchi M, Ueda K (2003) Intercalation activating fluorescence DNA probe and its application to homogeneous quantification of a target sequence by isothermal sequence amplification in a closed vessel. Anal Biochem 314:77–86PubMedCrossRefGoogle Scholar