Paul.xu at 18:58, 1 July 2016

2016-07-01T18:58:48Z

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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, <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,  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.

{|
|-
| align="CENTER" | '''pH''' 
| 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 
|}

 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 
| align="CENTER" | -51.2 
|}

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 
|}
[[Category:Protocols]]

Taq Purification Excellgen - Revision history

Paul.xu at 18:58, 1 July 2016