Editing Taq Muts (section)

== Purification of Taq-Muts, no tag<br> ==

1. Grow E. coli strain such as BL21(DE3) transformed with 353 pT7A3E-HC-TaqMuts in 8L LB/AMP at 37 °C.<br>2. Induce for 4 h with 1 mM IPTG.<br>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. <br>4. Thaw cells and homogenize.<br>5. Heat the lysate at 70 °C for 15 min.<br>6. Spin at 25000 rpm for 20 min, at 4 oC.<br>7. Measure volume, add saturatedor solid (NH4)2SO4 to 65% saturation<br>8. Incubate at 4 oC for 30 min.<br>9. Spin for 25000 rpm for 20 min, at 4 oC.<br>10. Resuspend pellet in '''Buffer 1'''

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

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

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

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

<br>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.<br>19. Load flow through and wash to a 15 ml phoshpate cellulose column.<br>20. Elute protein with '''Buffer 4'''.

<br>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

<br>21. Load to a 10 ml hydroxyapatite column (Bio-Rad), equilibrated with Buffer 4. <br>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. <br>23. Combine Muts fractions and dialyse against enzyme storage buffer. <br>



[[Category:Protocols]]