help: Problem encountered using GST fusion system - (Aug/21/2004 )

Hi, Dear All,
I am currrently using pGEX-2T vector to make some constructs. I've succeeded in protein expression. But I was in trouble with purification, since the fusion protein aggregates and forms inclusion bodies.... I used standard solubilization (8M urea, 1% Triton-X100) and typical refolding protocols (slow dilution and dialysis) to get my protein (about 32 KDa) out of the cell pellets...and fortunately, the refolded GST fusion protein bound to the beads and can also be cleaved by thrombin...however, there is almost nothing in the elution!!! and I found that the cleaved products were still bound to the beads....please help to explain these weired things....and give me some suggestion....

I've come accross various different protocols for elution of GST fusions, the most common are:

1. 10-50mM Glutothione in Tris pH 8

2. 20mM Glutothione in Tris pH 9

Try these and let me know.

Also, do you have any idea why a soluble GST fusion would not stick to beads?? I cannot get the protein to bind to 4B-Glutothione beads no matter what! I know it's soluble, I can see it on the gel, I resolubilised it like you did in 8M Urea, but it just wont stick. Any help would be appreciated.

I've come accross various different protocols for elution of GST fusions, the most common are:

1. 10-50mM Glutothione in Tris pH 8

2. 20mM Glutothione in Tris pH 9 (This method works really well now....almost 90% of the protein were eluted out from the beads)
Try these and let me know.

Also, do you have any idea why a soluble GST fusion would not stick to beads?? I cannot get the protein to bind to 4B-Glutothione beads no matter what! I know it's soluble, I can see it on the gel, I resolubilised it like you did in 8M Urea, but it just wont stick. Any help would be appreciated.

Maybe the protein you purified is not wellfolded (especially the GST part...). Or maybe the binding efficiency is way too low....to improve the efficiency, you can collect your supernatant and mix with the beads with S/N in falcon tube and put them on shaker for 1 or 2 hours for fully binding....according to some literature, addition of 1 mM DTT could improve the efficiency (i 've never try that)....And you should also check the pH....make sure it's in the right range...Hope these will help....

How did you do the refolding? The protein I refolded is inactivity!