FXD FF IEX - FXD GF - FXD UF - FXD 30Q IEX

Purification of recombinant P. f. Ferredoxin (HC3526)
Pyrococcus furiosus ferredoxin is a small electron transfer protein, which exist both in a form with a Fe3S4 center and a form with a Fe4S4 center. P.f. ferredoxin is extremely thermostable. The P.f. ferredoxin used in the experiment has been overexpressed recombinantly in E. coli.  For more information on this protein please consult rcsb.org

Buffers prepared for you:

Binding buffer: 3 l 20 mM Tris/HCl, pH 8.0

Elution buffer: 3 l 20 mM Tris/HCl, 1.0 M NaCl pH 8.0 

Gelfiltration buffer: 2 l 20 mM Tris/HCl, 0.15 M NaCl pH 8.0


Regeneration solutions:          
                        2 l Degassed Ultra-Pure water,

1 l 1 M NaOH.

1 l 2 M NaCl

2 l 20 % v/v ethanol.


Purification day one:

  1. Resuspend cells from one 650 mL culture with binding buffer to a volume of 40 ml.

  2. Prepare a 1 M sodiumdithionite by adding 0.087 g Na2S2O4 to 0.5 ml ultra-pure water.

  3. Add 80 µl 1 M sodiumdithionite to the cells to keep the protein on reduced form.

  4. Lyse the cells by sonication (in Satorius Labsonic P, 80% amplitude, 1 cycle) 3x30 sec on wet ice.

  5. Incubate the cells at 70°C for 10 min to denature most E. coli proteins, and then cool the mixture on ice.

  6. Centrifuge in high-speed centrifuge tubes at 4°C and 12000 rpm (≈18500 g) for 20 minutes.

  7. Prepare 120 ml 20 mM Tris/HCl pH: 8.0, 2 mM Na2S2O4 solution, by adding 0.042 g Na2S2O4 to 120 ml binding buffer.

  8. Add the supernatant to the freshly made buffer solution (point 7).

  9. Turn on ÄKTA Start, flush buffers, and attach a 16/10 Q Sepharose Fast Flow column.

  10. Start equilibration of the column (flow rate: 5.0 ml/min, pressure limit 0.50 MPa). 1 CV elution buffer followed by binding buffer until conductivity and UV have dropped and are stable.

  11. Load the protein solution onto the column (flow rate: 5.0 ml/min, pressure limit 0.50 MPa). Wash with 2 CV of binding buffer. Wash with a step gradient of 15 % elution buffer. Elute and collect the brown target protein with a step gradient of 40 % elution buffer.

  12. Wash the column for 5 CV with elution buffer. Reequilibrate with 5 CV of binding buffer.

  13. Store the protein in a 50 ml centrifuge tube in the refrigerator.


    Ultrafiltration:

  14. Reduce the volume of the protein solution by ultrafiltration using a stirred amicon cell with a PLBC 3000 MWCO membrane until the volume is app. 5 ml.

  15. Store the protein in a 15 ml centrifuge tube in the refrigerator.


    Equilibration of gel filtration column (the second class only):

  16. Flush buffers on ÄKTA Start using Ultra-Pure water as buffer B and gel filtration buffer as buffer A.

  17. Attach a 26/600 Superdex 75 pg Hiload column (flow rate: 1.0 ml/min, pressure limit 0.30 MPa).

  18. Fill the flask containing gel filtration buffer to maximum.

  19. Run method Gel start stop.


    Purification day two:

  20. Turn on ÄKTA Start, flush buffers, and attach a 26/600 Superdex 75 pg Hiload column.

  21. Filter the protein through a 0.45µm syringe filter.

  22. Load a maximum of 13 ml protein solution onto the column (flow rate: 2.6 ml/min, pressure limit 0.30 MPa). Elute and collect the brown target protein isocratically using 1 CV gel filtration buffer.

  23. Store the protein in a 50 ml centrifuge tube in the refrigerator.


    Reequilibration of gel filtration column (the second class only):

  24. Flush buffers on ÄKTA Start using Ultra-Pure water as buffer B and 20 % v/v ethanol as buffer A.

  25. Attach a 26/600 Superdex 75 pg Hiload column (flow rate: 1.0 ml/min, pressure limit 0.30 MPa).

  26. Fill the flask containing 20 % v/v ethanol to maximum.

  27. Run method Gel start stop.


    Purification day three:

  28. Turn on ÄKTA Start, flush buffers, and attach a 16/10 Source 30Q column.

  29. Start equilibration of the column (flow rate: 5.0 ml/min, pressure limit 0.50 MPa). 1 CV elution buffer followed by binding buffer until conductivity and UV has dropped and is stable.

  30. Dilute the protein solution 4 times with ultra-pure water.

  31. Filter the protein solution through a 0.45µm syringe filter.

  32. Load the protein onto the column (flow rate: 5.0 ml/min, pressure limit 0.50 MPa). Wash with 5 CV of binding buffer. Elute and collect the brown target protein with a linear gradient from 15% to 30% elution buffer over 20 CV.

  33. Wash the column for 5 CV with elution buffer. Reequilibrate with 5 CV of binding buffer.

  34. Determine the protein content of your solution in mg by UV-Vis on nanodrop.
    Mw: 7516 g/mol, ε408 nm = 17000 lmol-1cm-1.

  35. Store the protein in a 50 ml centrifuge tube in the freezer.


    Cleaning of Q Sepharose Fast Flow column:

  • 2 CV 2M NaCl,

  • 2 CV 1M NaOH,

  • 2-4 CV 2M NaCl [control for neutral pH with pH paper]

  • 2 CV ultra-pure water

  • 2 CV 20 % ethanol.







http://www.kemi.dtu.dk/Uddannelse/Elearning/Kursus26125_ChromatographicPurPro-Juni18/FXD_FF_IEX
18 NOVEMBER 2018