MBP-GFP AC

Purification of recombinant MBP-GFP (HC3446)

MBP-GFP is a recombinant fusion protein expressed in E. coli consisting of the maltose binding protein (MBP) and the green fluorescent protein (GFP). We will use this chimeric protein to illustrate and practice affinity chromatography and gel filtration.  MBP is the affinity-tag and GFP is a green protein that allows us to easily follow the protein through the purification process. 

Information on MBP can be found here:

wikipedia - maltose-binding protein

neb.com - maltose-binding-protein-expression

uniprot.org - P0AEX9

Information on GFP can be found here:

wikipedia - green fluorescent protein

proteopedia - green fluorescent protein

Buffers prepared for you:

Binding+gelfiltration buffer: 2.5 l 20mM Tris/H2SO4, 50mM (NH4)2SO4, pH 8.0

Elution buffer: 250 ml 20 mM Tris/H2SO4, 50mM (NH4)2SO4, 10mM maltose, pH 8.0

Regeneration solutions:         
                        1.5 l Degassed Ultra-Pure water,

0.2 l 0.5 M NaOH.

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

  3. Centrifuge the protein suspension at 4°C and 12000 rpm (≈18500 g) for 2x20 minutes and recover supernatant.

  4. Turn on ÄKTA Start, flush buffers, and attach a 16/10 Dextrin Sepharose column (flow rate: 1.0 ml/min).

  5. Start equilibration of the column (flow rate: 5.0 ml/min, pressure limit 0.30 MPa). 5 CV binding buffer. Check that UV is stable.

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

  7. Load the supernatant onto the column (flow rate: 5.0 ml/min, pressure limit 0.30 MPa). Wash with 5 CV of binding buffer. Elute and collect the target protein with 5 CV elution buffer.

  8. Reequilibrate with 5 CV of binding buffer.

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

  10. Regenerate column using 3 CV Ultra-Pure water, 3 CV 0.5 M NaOH, 4 CV Ultra-Pure water, check that pH is equal to or below 7, 3 CV 20 % v/v ethanol. Store column in refrigerator.

       

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

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

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

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

  14. Run method Gel start stop.


    Purification day two:

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

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

  17. 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 target protein isocratically using 1 CV gel filtration buffer.

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

       

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

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

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

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

  22. Run method Gel start stop.

       

    MBP-GFP

  23. MNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMEN
    AQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGLEVLFQGPGSEFRVPGSENLYFQGQFSKGEELFTGVVPILVELDGDVNGHKFSVSGEG
    EGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIT
    ADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKKLAAHHHHHHHH


    GFP

    GPGSEFRVPGSENLYFQGQFSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIS
    FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMV
    LLEFVTAAGITHGMDELYKKLAAHHHHHHHH

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