GST-Fusion Purification and Cleavage


PBS (1 L)

8 g       NaCl

0.2 g    KCl

1.44 g Na2HPO4

0.24 g  KH2PO4

- dissolve the above in 800 ml ddH2O, adjust pH to 7.3 with HCl, then bring to 1L


  1. Resuspend cells in 50 ml GST lysis buffer (PBS + 0.1% Triton X-100, 1 mM DTT, 5ug/ml DNAse I, 10 ug/ml RNAse A, 1 mg/ml lysozyme, and 1 mM PMSF)


  1. Incubate resuspended cells on ice for 30 minutes.


  1. Meanwhile, begin equilibrating a 5mL GST-Trap column (Amersham-Pharmacia) by pumping GST through it with a peristaltic pump.  Pump at least 3 column volumes (15 mL) through the column.


  1. Sonicate cells for one 20-second cycle.  (This should be enough sonication since we used lysozyme above.  Over-sonicating disrupts the GST tag and makes it so that the protein will not bind the column!


  1. Centrifuge at 15 000 rpm (JA-20 rotor) for 20 minutes.


  1. Collect supernantant, and load it very slowly on to the equilibrated GST-Trap column.  Pump at 0.5 mL/min or less.


  1. Wash column extensively with 150 mL PBS.  The pump can be run at more than 1 mL/min at this stage.


  1. Mix 40 units of thrombin in 5 mL PBS.  Pump this 5 mL thrombin solution in to the column, then shut down the pump.  Allow thrombin to cleave the fusion protein overnight at room temperature.  (If you have evidence that your protein degrades at room temperature, perform this step in the cold room).


  1. Elute you protein of interest with 4 x 5 mL fractions of PBS.  These fractions will contain your protein without GST attached.


  1. Elute GST from the column in a 15 mL fraction of GST elution buffer (50mM Tris-HCl, pH8.0 with 10 mM reduced glutathione).  This fraction will contain GST alone plus any GST-fusion that was not cleaved by thrombin above.


  1. Run gels of all fractions to determine purity, etc.  Pool and concentrated as desired.