Cleaved GST fusion protein retained on beads! - Affinity of GST-cleaved protein for glutathione sepharose (Mar/03/2005 )

I have successfully cleaved by protein of interest from it's GST-tag using PreScission protease (validated using a specific antibody on a western), following purification using glutathione sepharose. However I cannot elute my protein! It remains firmly bound to the glutathione sepharose beads. I have tried eluting with reduced glutathione up to 50mM (pH 8.0) and with various detergent addition (triton X-100, OGP, NP40, CHAPS, NDSB 256) and with high salt (500mM NaCl) or EDTA (1mM) - but with very little success. Adding detergent helps, but release of my recombinant protein is far from satisfactory. Please help if you can! Has anyone had problems with proteins minus the GST-tag sticking to glutathione sepharose? It appears that non-specific hydrophobic interactions are preventing solubilisation and/or elution from the beads whether the GST-tag is present or not.

I'm having the same problem as you! After cleavage (which is 100%), about 90% remains stuck on the beads. I have also tried everything you have tried, and have had very little success. In desperation, I have tried to elute the GST-fusion protein, using pretty much the same methods, and going up to 50mM reduced glutathione. Again, about 90% remains stuck to the beads.
It's reassuring to know that I am not the only one with this problem, and I would really appreciate any hints to solve it!

I have not worked with GST tags, but used glutathone sepharose for other glutathione binding protein.

I do not under when you have removed the GST tag. GST tag is to facilitate purification of the recombinant protein by affinity chromatography with glutathione sepharose. The tag is removed after purification.

Kindly tell me know if you are facing problem with eluting GST fusion protein or with the tag-deleted protein.

If you have removed the tag before loading onto the column, then the mode of binding might be other than bioaffinity. GST is the moiety which has affinity for glutathione and consequently confers the fusion protein with affinity for glutathione.

I use both the GST-fusion and GST-tag removed protein.

For the GST-fusion, binding to the beads is fine, but elution with reduced glutathione is a major problem. At most, I only ever see about 20% being eluted (even in 40mM reduced glutathione). I have tried changing NaCl concentration, adding DTT, detergents, etc and nothing helps. I would appreciate any suggestions.

For cleaved protein, I bind the GST-fusion to the beads, cleave overnight at 4C with Prescission protease, then remove the supernatant (which should be the cleaved protein - GST tag). My cleavage is usually 100%, but the cleaved protein (approx. 90%) remains stuck to the beads. Again, I have tried adding detergents, DTT, increasing NaCl concentration, etc, but nothing helps. Again, any suggestions would be appreciated!

I have done the same as you! I only elute approx 10% of the GST fusion protein from glutathione sepharose beads. After GST tag cleavage with PreScission protease and as you say, the cleaved protein of interest should remain in the supernatant - free from the beads, but alas the protein must have its own natural affinity for glutathione sepharose. Since I have tried detergents, increasing salt conc etc (as detailed above), I have decided to incorporate a x6 His-tag into my vector just before the PreScission Protease site. I will then use x6 His as my tag for purification using Ni-NTA beads. Apparently GST helps to make proteins more soluble, so I am going to leave the GST on my fusion protein. Since the PreScission Protease site if after the GST and x6 His I will remove both tags. Novagen produces HRV 3C protease which is essentially the protease used in Amersham's PreScission Protease but it binds to Ni-NTA and thus can be used in the same way, but making use of a different affinity tag. This is the only way I can think of curing this problem. What do you think?

Hello,
I am also facing same type of the problem. i have also expressed my protein it binds with sepharose good i cleaved it by Prescission protease. but when eluted with Prescission buffer it is not eluting. but when i eluted it with glutathione reduced then it elutes with GST tag. i tried to elute my protein by increasing salt conc but not helpful. if any one have suggestions abt the problem kindly mail me soon. i will be thankful for that.

BS, Germany

This might work, as I have heard it from several people but have not tried it myself. Pre-incubate the Glutathione beads with an E.coli extract (not overexpressing your protein of interest). Then wash the beads several times, and use those beads for purifying the GST tagged protein. This will use up all the available sites for hydrophobic interactions, only allowing for specific interactions. Also, using lesser amounts of beads might help.

Best wishes.

As someone has already said it seems your protein has an affinity for the glutathione. Have you considered just eluting the fusion protein, cutting it and then purifiying tag from protein on another column? You could also try running a denatureant onto the column which will cause the proteins to come off the column unfolded. The biggest problem with that is if your protein can refold on its own.