Comparison of the thermal stabilities of the αβ heterodimer and the α subunit of avian myeloblastosis virus reverse transcriptase.
ABSTRACT Avian myeloblastosis virus reverse transcriptase (AMV RT) is a heterodimer consisting of a 63-kDa α subunit and a 95-kDa β subunit. In this study, we explored the role of the interaction between the α and β subunits on AMV RT stability. The recombinant AMV RT α subunit was expressed in insect cells and purified. It exhibited lower thermal stability than the native AMV RT αβ heterodimer. Unlike the αβ heterodimer, the α subunit was not stabilized by template-primer. These results suggest that interaction between the α and β subunits is important for AMV RT stability.
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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.Biotechnology Letters 03/2012; 34(7):1209-15. · 1.85 Impact Factor