Taq Purification Excellgen
1. On Friday, Use pipet tip to get tiny amout of E coli glycerol stcok and inoculate in 15 ml LB/K/Cm medium, grow o/n at 32 oC. The 15 ml culture is called starter stock
2038: pL42-sso7d-KlenTaqsso7d fuse to Klen Taq, C6XHis
2324: pL6H-sso7d-wt-Taq full length wt Taq fused to N-sso7d
2. On Saturday before 5 PM, transfer ~3 ml of starter stock into 500 ml, LB/K/Cm medium, grow o/n at 32 oC. Store the rest of starter stock in 4 oC fridge.
3. Sunday afternoon: split 500 ml medium into 8 flasks of 1000 ml LB/K/Cm, grow to OD600 ~ 0.8~1.2, grow o/n at 32 oC
4. Monday morning adjust to 42 oC, induce for 4~6 hours.
5. Spin down cells, store at -80 oC.
6. Add ~400 ml Buffer A, use a blender to break the cells into a suspension
Buffer A: 25 mM Tris-HCl, pH 7.9~8.0, 0.1 mM EDTA, 500 mM KCl or NaCl, 0.5% Tween 20, 1 mM b-ME
7. Put into 75 oC water bath, inclubate for 30 min. (split into 200 ml aliquots to allow more rapid temperature equilibration).
8. Cool on ice. Spin at 6000 rpm for 10~15 min using a Sorvall GS3 rotor at 4 oC. Decant and collect supernatant. Remove floating stuff using cheese-cloth or coffer filter paper if necessary. Discard cell debris and denatured protein pellet.
9. Load the supernatant to a column with 10~20 ml Ni beads (or mix with beads at 4 oC o/n).
Ni Washing Buffer: 25 mM Tris-HCl, pH 7.9~8.0, 0.1 mM EDTA, 500 mM NaCl, 0.5% Tween 20, 20 mM Imidazole, 1 mM b-ME
10. Wash column with Ni Washing Buffer until the OD280 value in eluate has not much protein (OD280 <0.1), usually washing with 100~150 ml of the Ni Washing Buffer
11. Elute protein with Buffer B,
Buffer B: 25 mM Tris-HCl, pH 7.9~8.0, 500 mM NaCl, 150 mM imidazole, 1 mM b-ME, 0.5% Tween 20
12. Pool the fractions containing the peak of protein
13. Dialyse o/n against 2 liter of Buffer C (20 mM Tris-HCl, pH 8.8, 50 mM NaCl)
14. Load dialysed Taq to a Q HP column, pre-equilibrated with Buffer C (20 mM Tris-HCl, pH 8.8, 50 mM NaCl).
make sure to wash dialyse tube with Buffer C to minimize the lose of Taq protein.
Note:
If it is urgent, Taq protein on Ni column can be eluted using Low Salt Buffer B (25 mM Tris-HCl, pH 7.9~8.0, 100 mM NaCl, 150 mM imidazole, 1 mM b-ME, 0.5% Tween 20), then dilute the Taq protein with equal volume of a buffer without NaCl (20 mM Tris-HCl, pH 8.3) before loading to Q HP column.
15. Wash column with Buffer C (20 mM Tris-HCl, pH 8.3, 50 mM NaCl) until OD280 reach baseline, usually 5~10 X column volume
16. Elute protein with a linear gradient of 50 mM to 1 M NaCl using Buffer C and Buffer D (20 mM Tris-HCl, pH 8.3, 1 M NaCl), usually 5~10 column volume (CV). If protein has not been eluted out from column, use 2~5 CV of Buffer D to elute the protein.
17. Wash Q HP column with Buffer C, wash FPLC system with ddH2O
18. Combine fractions containing pure Taq protein. Measure OD280 and QC for enzyme activity by PCR.
19. Dialyse vs 2 changes of 50 ml of Storage Buffer (20 mM HEPES, pH 7.2, 100 mM KCl, 1 mM dithiothreitol, 50% glycerol).
20. Store dialysed enzyme at -80 oC.
| pH |
Charge |
| 4.00 | 80.9 |
| 4.50 | 55.6 |
| 5.00 | 33.0 |
| 5.50 | 20.3 |
| 6.00 | 13.4 |
| 6.50 | 7.7 |
| 7.00 | 2.9 |
| 7.50 | 0.1 |
| 8.00 | -1.5 |
| 8.50 | -3.2 |
| 9.00 | -6.5 |
| 9.50 | -14.4 |
| 10.00 | -28.0 |
Estimated charge of wt Taq (pI =6.20) over pH range, no his tag
| pH | Charge |
| 4.00 | 100.2 |
| 4.50 | 63.7 |
| 5.00 | 31.1 |
| 5.50 | 12.9 |
| 6.00 | 3.2 |
| 6.50 | -4.5 |
| 7.00 | -10.8 |
| 7.50 | -14.4 |
| 8.00 | -16.5 |
| 8.50 | -18.6 |
| 9.00 | -23.0 |
| 9.50 | -33.3 |
| 10.00 |
-51.2 |
Estimated charge of sso7d-wt-Taq (pI = 6.71), N- his tag
| pH | Charge |
| 4.00 | 119.7 |
| 4.50 | 80.0 |
| 5.00 | 44.3 |
| 5.50 | 24.2 |
| 6.00 | 13.0 |
| 6.50 | 3.6 |
| 7.00 | -4.3 |
| 7.50 | -8.9 |
| 8.00 | -11.5 |
| 8.50 | -14.1 |
| 9.00 | -19.5 |
| 9.50 | -32.4 |
| 10.00 | -54.7 |