Purification of GST fusion proteins in E.coli GST融合蛋白纯化（方法四）筛选表达

Pick several colonies of E.coli transformed with the pGEX recombinants into separate tubes containing 2 ml of 2 x YTA medium.

Grow o/n at 20~37?C.

Add 2 ml culture into 100 ml 2 x YTA medium.

Grow liquid cultures to an A₆₀₀ of 0.6-0.8 (about 2 h) with vigorous agitation at 20~37?C.

Incubate fusion protein expression by adding 50 μl of 100 mM IPTG (final concentration 0.1 mM).

Continue incubation for an additional 1~2 h

Transfer the liquid cultures to 50 ml tubes.

Centrifuge at 3000 rpm for 10 min at 4?C and discard the supernatant.

Resuspend each pellet in 5 ml of ice-cold 1 x PBS.

Sonicate on ice with three to five brief (10 sec) pulse.

Transfer to new microcentrifuge tubes (1 ml, each) and centrifuge for 10 min at 10000 rpm at 4?C to remove insoluble materials. Transfer the supernatants to fresh tubes.

Add 50 μl of a 50% slurry of Glutathione Sepharose 4B to each supernatant and rock gently for 10 min at 4?C.

Add 1 ml of 1 x PBS to each tube, vortex briefly, and centrifuge 10000 rpm at for 5 sec to sediment the Sepharose beads.

Discard sup, repeat 1 x PBS wash twice.

Elute the fusion protein by adding 100 μl of Glutathione Elution Buffer. Suspend the Sepharose beads and incubate for 10 min at 4?C.

Centrifuge in a microcentrifuge for 5 min to sediment the Sepharose beads, then transfer the supernatant to fresh tubes.