GST fusion recombinant antigens degradation

[paramyosin]

I have purified several recombinant proteins fused to GST and I always have seen a common degradation which leaves GST (approx. 25 kDa) alone and other bands. I have tryed to avoid this degradation by adding protease inhibitors, PMSF, EDTA...but I did to improve the situation.

Any hint? does someone realize of something similar?

Thank you!!

[relaxin]

Did you use the right type of protease inhibitor cocktail for your cells? The correct concentration?

[paramyosin]

I think so, I use the EDTA free from Roche, the preparation should be 1x from a 50x stock....

I have seen old posts regarding this same problem and there are several cases!. It seems to be a frequent degradation of proteins fused to GST, at my knoledge there is a problem with one of the commercial tags which are between the ORF of GST and the polilinker, it should be very sensible to the proteases action and not very specific of course, maybe, eliminating this target from the original construction it would be possible to avoid this situation. I have the target of trombin and of Factor Xa.
What do you think?

[relaxin]

It could be a possibility. I have no experience with GST fusion protein. I wonder if specific inhibitors of trombin and of Factor Xa are available.

[paramyosin]

Actually, I think that PMSF is a thrombin inhibitor and I am using it. I have tryed to resupend the cell pellet once the culture is centrifuged in buffer with 6 M urea in order to desnaturalize all the proteases in the clarified extract, and EVEN in this case I obtain degradation, I wonder if the degradation occurs during the culture growing. I am using a BL21 which is ompT as it is sugested for these fusion proteins to avoid degradation.
I am using everything esterilized...

[CrowSan]

You may have to live with this. I have seen it with various tags (inc GFP). I am guessing the linker is more solvent exposed (especially if it contains a row of glycines) making it suscpetable to intracellular proteases (many of which in general don't really have specific recognition sites as such but will chomp on anything). The degradation probably happens inside the cell prior to lysis (but as I haven't really checked this I can't say for sure).