Taq Muts

Purification of Taq-Muts, no tag

1. Grow E. coli strain such as BL21(DE3) transformed with 353 pT7A3E-HC-TaqMuts in 8L LB/AMP at 37 °C.
2. Induce for 4 h with 1 mM IPTG.
3. Spin down cells, resupend cells in 20 mM KPO4 (pH 8.0), 100 mM NaCl, 1 mM beta-ME, 0.1 mM EDTA, store cells at -20 or -80 °C.
4. Thaw cells and homogenize.
5. Heat the lysate at 70 °C for 15 min.
6. Spin at 25000 rpm for 20 min, at 4 oC.
7. Measure volume, add saturatedor solid (NH4)2SO4 to 65% saturation
8. Incubate at 4 oC for 30 min.
9. Spin for 25000 rpm for 20 min, at 4 oC.
10. Resuspend pellet in Buffer 1

20 mM potassium phosphate, pH 8.0, 100 mM NaCl, 5% (v/v) glycerol, 1 mM beta-ME 0.1 mM EDTA

11. Desalt the protein solution on a Sephedex G25 column using Buffer 1.
12. Load to 20 ml QFF column.
13. Wash column with Buffer 1
14. Elute with linear gradient from Buffer 1 to Buffer 2

20 mM potassium phosphate, pH 8.0, 1 M mM NaCl, 5% (v/v) glycerol, 1 mM beta-ME 0.1 mM EDTA

15. MutS will be eluted at ~ 260 mM NaCl.
16. Combine fractions containing MutS.
17. Dilue protein solution with Buffer 3

20 mM Tris-HCl or HEPES, pH 7.8, 50 mM NaCl, 5% (v/v) glycerol, 0.1 mM EDTA, 1 mM beta-ME

18. Load to a 15 ml SP FF column, collect flow through and wash.
19. Load flow through and wash to a 15 ml phoshpate cellulose column.
20. Elute protein with Buffer 4.

50 mM Tris-HCl or 20 mM HEPES, pH 7.8, 0.4 M NaCl, 5% (v/v) glycerol, 0.1 mM EDTA, 1 mM beta-ME

21. Load to a 10 ml hydroxyapatite column (Bio-Rad), equilibrated with Buffer 4.
22. Elute protein using a 60-ml linear gradient of 0 –120 mM potassium phosphate in HEPES buffer. MutS eluted at 60 mM potassium phosphate.
23. Combine Muts fractions and dialyse against enzyme storage buffer.