Purification of recombinant A. v. Plastocyanin (HC3508)

Plastocyanin

Plastocyanin is a small blue copper protein transferring electrons in the photosynthesis machinery. Plastocyanin (PCu) purified in this project is from the cyanobacterium Anabaena variabilis and is recombinantly expressed in E. coli, where it is located in the periplasm. More information on A. v. PCu can be found here. 

Buffers prepared for you:

Resuspension solution: 40 ml 0.5 mM MgCl2

Incubation solution: 1 ml 100 mM CuSO4

Binding buffer: 2 l 5 mM MES/NaOH, pH 6.5

Buffers you need to prepare:

Elution buffer: 1000 ml 5 mM MES/NaOH, 0.5 M NaCl, pH 6.5

Prepared in advance by laboratory technician:

  1. One tube of cells from 650 ml culture is frozen and thawed three times. Freeze for 15 minutes at
    - 80 °C and thaw for 15 minutes on water bath (room temperature) for each cycle.


    Before buffer preparation:

  2. The cells is resuspended in 40 ml 0.5 mM MgCl2 by gentle swirling using a glass rod and then incubate on wet ice for 25 minutes with occasional stirring.

  3. Centrifuge in high-speed centrifuge tubes at 4°C and 12000 rpm (≈18500 g) for 25 minutes.

  4. Retain supernatant and add 0.2 ml 100 mM CuSO4, incubate for 10 minutes at 4°C. Repeat three times thus giving a final concentration of 2.0 mM CuSO4.

  5. Incubate until purification.


    Purification day one:

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

  7. 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 the conductivity and UV have dropped and are stable.

  8. Centrifuge the protein suspension 4°C and 12000 rpm (≈18500 g) for 20 minutes.

  9. Check and adjust conductivity of supernatant to below 250 µS/cm by dilution with Ultra-Pure water. Keep on ice until use.

  10. Load the supernatant 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 blue target protein with a step gradient of 13 % elution buffer.

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

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


    Purification day two:

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

  14. 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.

  15. Check and adjust conductivity of supernatant to below 300 µS/cm by dilution with Ultra-Pure water.

  16. Filter the supernatant through a 0.45µm syringe filter.

  17. Load the supernatant 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 blue target protein with a linear gradient from 0.4 % to 15 % elution buffer over 20 CV.

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

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


http://www.kemi.dtu.dk/Uddannelse/Elearning/Kursus26125_ChromatographicPurPro-Juni18/IEXplastocyanin
22 SEPTEMBER 2018