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
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)
resuspended cells on ice for 30 minutes.
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.
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!
at 15 000 rpm (JA-20 rotor) for 20 minutes.
supernantant, and load it very
slowly on to the equilibrated GST-Trap column. Pump at 0.5 mL/min or less.
column extensively with 150 mL PBS.
The pump can be run at more than 1 mL/min at this stage.
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).
you protein of interest with 4 x 5 mL fractions of PBS. These
fractions will contain your protein without GST attached.
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.
gels of all fractions to determine purity, etc. Pool and concentrated as desired.