Purification of GST-tagged Proteins

Transformation:

Day 1:

Transform expression plasmid into BL21/DE3 pRI952.

Plate on LBA (Amp) plates.

Growth and induction:

Day 2:

Start 10 ml over night culture in LB w. 100 µg/ml Amp, 50 µg/ml Chloramp.

Day 3:

Dilute culture 1:100 into 1000 ml LB w. 100 µg/ml Amp, 50 µg/ml Chloramp. (prewarmed is better).

Follow A600.

At A600 between 0.4-0.5 (do not overgrow!!) transfer culture to 15C.

After 10-15 min, add IPTG to 0.2 mM final concentration.

Grow over night at 15C.

(Temperature, time and IPTG concentration may have to be optimized for individual proteins).

Purification:

Day 4:

Pellet cells at 5,000 rpm, 8 min.

Resuspend cells completely (!) in 25 ml ice cold TKET w. 1 mM PMSF (PMSF freshly added, TOXIC!).

(PMSF has a short half-life in aqueous solutions).

Sonicate 8x30'' with 30'' intervals at 4.5 amplitude on ice.

Add Triton X-100 to 0.5% final.

Save 50 µl for analysis.

Nutate at 4C for 15 min.

Spin 11,000 rpm, 15 min.

For each prep aliquot 1.5 ml Glutathione Sepharose slurry and wash 2 times with 10 ml TKET (1,000 rpm, 1 min). (The amount of Glutathione Sepharose may have to be titrated).

Add supernatant from sonicated cells to washed Glutathione Sepharose beads. (Supernatant should be yellow, avoid pellet!).

Nutate 1-2 hours at 4C.

Spin 1,000 rpm 1 min.

Remove and save supernatant. (Transfer 50 µl to tube for analysis, store remainder at -80C in case more protein needs purified).

Wash beads 5 times with 10 ml TKET.

After final wash, remove most of the buffer.

Elution:

Add 1 ml 100 mM Tris-HCl pH7.5, 100 mM KCl, 20 mM glutathione.

Nutate at RT, 15 min.

Spin 1,000 rpm, 1 min.

Transfer supernatant to tube. Test protein concentration by Bradford (below).

Redo elution until little protein comes off beads (usually 2-3 times).

Pool eluates and dialyse against PBS or other buffer at 4C.

Day 5:

Aliquot dialysed proteins to tubes.

Measure concentration by Bradford and analyse 2-4 µg on SDS-PAGE.

Bradford:

Mix

800 µl water

200 µl Bio-Rad protein assay concentrate

2-20 µg protein

Leave at RT 5 min

Measure A595 (use standard w/o protein)

Compare to a BSA titration from 2-20 µg

Buffers:

TKET:

10 mM Tris-HCl pH 7.5

100 mM KCl

0.1 mM EDTA

0.05 % Triton X100