Figures & data
Fig. 1. Structure of MMLV RT Variant L435K.
Note: The structure is based on Protein Data Bank No. 1RW3. (A) Overall structure. (B) Close-up view of the surface region, in which Phe303 and Leu304 are located. (C) Close-up view of the surface region, in which Leu432, Val433, and Ile434 are located.
Fig. 2. Analysis of Single MMLV RT Variants.
Note: (A) SDS-PAGE under reducing conditions. Coomassie Brilliant Blue-stained 10% SDS-polyacrlamide gel is shown. The arrow indicates the band corresponding to MMLV RT. (B) Specific activity. One unit was defined as the amount that incorporates 1 nmol of dTTP into poly(rA)-p(dT)15 in 10 min. Relative specific activity was defined as the ratio of the specific activity of RT to that of WT (34,000 units/mg). (C) Thermal stability. RT at 50 nM was incubated in 10 mM phosphate buffer (pH 7.6), 2 mM DTT, 0.2% v/v Triton X-100, 10% v/v glycerol, and 28 μM poly(rA)-p(dT)15 at 48 or 50 °C for 10 min. Then a dTTP incorporation reaction was carried out at 37 °C. Relative activity was defined as the ratio of the initial reaction rate of RT with 10 min incubation at 48 or 50 °C to that without the incubation.
Fig. 3. Thermal Stability of Multiple MMLV RT Variants.
Note: RT at 50 nM was incubated with 28 μM poly(rA)-p(dT)15 at 50ºC for 10 min. Then a dTTP incorporation reaction was carried out at 37 °C. Relative activity was defined as the ratio of the initial reaction rate of RT with 10 min incubation at 50 °C to that without incubation.
