GST Fusion Protein Purification

  1. Select a single bacterial colony and grow at 37oC overnight in 100 ml LB containing 50 ug/ml ampicillin.

  2. Inoculate 1 liter LB containing 50 ug/ml ampicillin with 20 ml overnight culture.

  3. Incubate at 30oC until the O.D.(600) equals 0.5 to 0.6, approximately 1-2 hours.

  4. Add IPTG to 0.5 mM and incubate for 1-16 hours at 30oC. The optimal length of induction should be predetermined in smaller culture prior to this step.

  5. Harvest cells by centrifugation at 10,000 x g for 10 minutes.

  6. Decant supernatant. Resuspend in 20 ml BPER (Pierce Corp) plus proteasome inhibitors, making sure suspension is homogeneous. Once homogeneous, shake for 10 minutes.

  7. (Optional) Sonicate 6 x 15 seconds to reduce viscosity.

  8. Save a 1 ml aliquot. Separate soluble proteins from insoluble material by centrifugation at 20,000 x g for 15 minutes.

  9. Collect supernatant into a new tube, and save a 1 ml aliquot.

  10. Load supernatant onto a 5 ml GST column prewashed with lysis buffer.

  11. After loading, wash column with 20 ml lysis buffer.

  12. Wash column with 20 ml wash buffer.

  13. Elute proteins bound to column with 20ml wash buffer plus 10 mM glutathione.

  14. Determine fractions containing appropriate purified protein by Western blotting, Bradford assay, and Coomassie staining.
Note that protease inhibitors are added from stocks to give 50 fold dilution.
Example: 20 ul of stock per 1ml buffer.

Use IMMEDIATELY after adding protease inhibitors.


LYSIS BUFFER
(20 mM Tris-HCL, pH 8.0, 200 mM NaCl, 1 mM EDTA, 1 mM EGTA)

TO MAKE 500 ml:

  • 10 ml 1 M Tris, pH 8 stock solution
  • 5.8 g NaCl
  • 1.0 ml 0.5 M EGTA stock solution
  • 1.0 ml 0.5 M EDTA stock solution

    WASH BUFFER
    (20 mM HEPES-KOH, pH 7.6, 350 mM NaCl, 1 mM DTT)

    TO MAKE 500 ml:

    • 10 ml 1 M HEPES-KOH, pH 7.6 stock solution
    • 10.15 g NaCl
    • 77 mg DTT

    50X STOCK Protease Inhibitors (store at -20oC)

    PMSF (87 mg/ml)
    ([500 mM] phenylmethylsulfonyl fluoride)

    TO MAKE 30 ml:  Dissolve 2.61g PMSF in DMSO to equal a final volume of 30 ml.

    LEUPEPTIN AND PEPSTATIN (5 mg/ml)

    TO MAKE 10 ml:  Dissolve 50 mg LEUPEPTIN or PEPSTATIN in DMSO to equal a final volume of 10 ml.

    TPCK (5 mg/ml)
    (tosylphenylalanine chloromethyl ketone)

    TO MAKE 10 ml:  Dissolve 50 mg TPCK in DMSO to equal a final volume of 10 ml.


    * Note: The EDTA in the Lysis buffer is also a protease inhibitor.