Figure 9
Evaluation of temperature, pH and salt concentration effect on TsoI REase activity. (A) The optimum temperature range of TsoI REase. 0.5 μg of T7 DNA (= 0.02 pmol recognition sites) was digested with 1 pmol (0.05 u) of TsoI in standard buffer supplemented with 50 μM SAM for 30 min in the temperature range from 45 to 65°C. Lane M1, GeneRuler™ 1 kb DNA Ladder (Thermo Fisher Scientific (Fermentas); selected bands marked); lane K, undigested T7 DNA; lane 1, 45°C; lane 2, 50°C; lane 3, 55°C; lane 4, 60°C; lane 5, 65°C; lane M2, GeneRuler™ 100 bp Plus DNA Ladder (Fermentas), selected bands marked. (B) The pH activity range of TsoI REase. 0.3 μg (= 0.6 pmol recognition sites) single site PCR substrate (390 bp) was digested with 0.33 pmol TsoI (0.016 u) in the pH range from 4.0 to 10.5 for 30 min at 55°C as described in the Methods section. GeneRuler™ 100 bp Plus DNA Ladder (Thermo Fisher Scientific (Fermentas); selected bands marked); lane K, undigested PCR fragment; lanes 1–14, PCR fragments after incubation with wt TsoI at indicated pH values: lane 1, at pH 4.0; lane 2, 4.5; lane 3, 5.0; lane 4, 5.5; lane 5, 6.0; lane 6, 6.5; lane 7, pH 7.0; lane 8, 7.5; lane 9, 8.0; lane 10, 8.5; lane 11, 9.0; lane 12, 9.5; lane 13, 10.0; lane 14, 10.5. (C) The influence of ionic strength on TsoI REase activity. 0.3 μg (= 0.6 pmol recognition sites) single site PCR substrate (390 bp) was digested with 0.33 pmol TsoI (0.016 u) in the NaCl concentration range from 0 to 60 mM for 30 min at 55°C in the standard reaction buffer, devoid of initial NaCl content: 10 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 0.01 mg/ml BSA, 0.5 mM DTT, 50 μM SAM. Lane M, GeneRulerTM 100 bp Plus DNA Ladder (Thermo Fisher Scientific (Fermentas); selected bands marked); lane K, undigested PCR fragment; lanes 1–7, digested PCR fragment: lane 1, without NaCl; lane 2, with 10 mM; lane 3, 20 mM; lane 4, 30 mM; lane 5, 40 mM; lane 6, 50 mM; lane 7, 60 mM.