Editing Taq Purification Excellgen

1. On Friday, Use pipet tip to get tiny amout of E coli glycerol stcok and inoculate&nbsp; 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&nbsp;&nbsp; &nbsp;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, <s>grow to OD600 ~ 0.8~1.2</s>, 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,&nbsp; 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.&nbsp; (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,&nbsp; 0.1 mM EDTA, &nbsp; 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,&nbsp; 150 mM imidazole, 1 mM b-ME, 0.5% Tween 20

12. Pool the fractions containing the peak of protein

13. Dialyse&nbsp;<span style="line-height: 20.8px;">o/n</span><span style="line-height: 20.8px;">&nbsp;against</span><span style="line-height: 20.8px;">&nbsp;2</span><span style="line-height: 20.8px;">&nbsp;liter of '''Buffer C''' (20 mM Tris-HCl, pH 8.8, 50 mM NaCl)</span>

14. Load dialysed &nbsp;Taq to a Q HP column, pre-equilibrated with&nbsp;'''Buffer C'''<span style="line-height: 20.8px;">&nbsp;(20 mM Tris-HCl, pH 8.8, 50 mM NaCl).</span>

<span style="line-height: 20.8px;">make sure to wash dialyse tube with Buffer C &nbsp;to minimize the lose of Taq protein.</span>

Note:

If it is urgent, Taq protein on Ni column can be eluted using&nbsp;'''Low Salt Buffer B'''&nbsp;(<span style="line-height: 20.8px;">25 mM Tris-HCl, pH 7.9~8.0, '''100 mM NaCl''',&nbsp; 150 mM imidazole, 1 mM b-ME, 0.5% Tween 20), then dilute the Taq protein with </span>'''&nbsp;equal volume of a&nbsp;buffer without NaCl ('''<span style="line-height: 20.8px;">20 mM Tris-HCl, pH 8.3) before loading to Q HP column.</span>



<span style="line-height: 20.8px;">15. Wash column with &nbsp;</span>'''Buffer C ('''<span style="line-height: 20.8px;">20 mM Tris-HCl, pH 8.3, 50 mM NaCl) until OD280 reach baseline, usually 5~10 X column volume</span>

<span style="line-height: 20.8px;">16. Elute protein with a linear gradient of 50 mM to 1 M NaCl using '''Buffer C''' and '''Buffer D'''&nbsp;(20 mM Tris-HCl, pH 8.3, '''1&nbsp;M '''NaCl), usually 5~10 column volume (CV). If protein has not&nbsp;been eluted out from column, use 2~5 CV of&nbsp;</span>'''Buffer D&nbsp;'''to elute the protein.

17. Wash Q HP column with&nbsp;'''Buffer C, '''wash FPLC system with ddH<sub>2</sub>O

18. Combine fractions containing pure Taq protein. Measure OD280 and QC for enzyme activity by PCR.

19. Dialyse&nbsp;vs 2 changes of 50 ml of&nbsp;&nbsp;'''Storage Buffer''' (20 mM HEPES, pH 7.2, 100 mM KCl, 1 mM dithiothreitol, 50% glycerol).

20. Store dialysed enzyme&nbsp;at -80 oC.



{|
|-
| align="CENTER" | '''pH'''<br/>
| align="CENTER" | '''Charge'''
|-
| align="CENTER" | 4.00
| align="CENTER" | 80.9
|-
| align="CENTER" | 4.50
| align="CENTER" | 55.6
|-
| align="CENTER" | 5.00
| align="CENTER" | 33.0
|-
| align="CENTER" | 5.50
| align="CENTER" | 20.3
|-
| align="CENTER" | 6.00
| align="CENTER" | 13.4
|-
| align="CENTER" | 6.50
| align="CENTER" | 7.7
|-
| align="CENTER" | 7.00
| align="CENTER" | 2.9
|-
| align="CENTER" | 7.50
| align="CENTER" | 0.1
|-
| align="CENTER" | 8.00
| align="CENTER" | -1.5
|-
| align="CENTER" | 8.50
| align="CENTER" | -3.2
|-
| align="CENTER" | 9.00
| align="CENTER" | -6.5
|-
| align="CENTER" | 9.50
| align="CENTER" | -14.4
|-
| align="CENTER" | 10.00
| align="CENTER" | -28.0<br/>
|}

<br/>Estimated charge of wt Taq (pI =6.20) over pH range, no his tag

{|
|-
| align="CENTER" | '''pH'''
| align="CENTER" | '''Charge'''
|-
| align="CENTER" | 4.00
| align="CENTER" | 100.2
|-
| align="CENTER" | 4.50
| align="CENTER" | 63.7
|-
| align="CENTER" | 5.00
| align="CENTER" | 31.1
|-
| align="CENTER" | 5.50
| align="CENTER" | 12.9
|-
| align="CENTER" | 6.00
| align="CENTER" | 3.2
|-
| align="CENTER" | 6.50
| align="CENTER" | -4.5
|-
| align="CENTER" | 7.00
| align="CENTER" | -10.8
|-
| align="CENTER" | 7.50
| align="CENTER" | -14.4
|-
| align="CENTER" | 8.00
| align="CENTER" | -16.5
|-
| align="CENTER" | 8.50
| align="CENTER" | -18.6
|-
| align="CENTER" | 9.00
| align="CENTER" | -23.0
|-
| align="CENTER" | 9.50
| align="CENTER" | -33.3
|-
| align="CENTER" | 10.00<br/>
| align="CENTER" | -51.2<br/>
|}

Estimated charge of sso7d-wt-Taq (pI = 6.71), N- his tag

{|
|-
| align="CENTER" | '''pH'''
| align="CENTER" | '''Charge'''
|-
| align="CENTER" | 4.00
| align="CENTER" | 119.7
|-
| align="CENTER" | 4.50
| align="CENTER" | 80.0
|-
| align="CENTER" | 5.00
| align="CENTER" | 44.3
|-
| align="CENTER" | 5.50
| align="CENTER" | 24.2
|-
| align="CENTER" | 6.00
| align="CENTER" | 13.0
|-
| align="CENTER" | 6.50
| align="CENTER" | 3.6
|-
| align="CENTER" | 7.00
| align="CENTER" | -4.3
|-
| align="CENTER" | 7.50
| align="CENTER" | -8.9
|-
| align="CENTER" | 8.00
| align="CENTER" | -11.5
|-
| align="CENTER" | 8.50
| align="CENTER" | -14.1
|-
| align="CENTER" | 9.00
| align="CENTER" | -19.5
|-
| align="CENTER" | 9.50
| align="CENTER" | -32.4
|-
| align="CENTER" | 10.00
| align="CENTER" | -54.7<br/>
|}
[[Category:Protocols]]