Cleaved GST fusion protein retained on beads!
Posted 03 March 2005 - 09:14 AM
Posted 04 March 2005 - 07:21 AM
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!
Posted 06 March 2005 - 10:36 PM
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.
Posted 07 March 2005 - 03:25 AM
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!
Posted 08 March 2005 - 09:50 AM
Posted 26 January 2011 - 07:29 AM
Here is what I do finally after trying the things you already mentioned:
-express the GST tagged protein from pGEX6p1 plasmid
-bind to GST beads
-elute with reduced gluthathione
-Test elution fractions with dot test (on a membrane)
-collect and pool the fractions containing protein
-Incubate o/n at 4C (at this step cleavage is 100% successful)
-Incubate the digest-mix with GST beads
- collect the supernatant
-purify over S75 coloumn
GST is still there and in big amounts compared to my protein of interest
Reminder: yes i tried digesting the protein on the beads but no way it is coming off the beads..
Thanks in advance
Posted 12 February 2011 - 11:30 AM
Edited by vivekp, 12 February 2011 - 11:32 AM.
Posted 24 February 2011 - 02:58 AM
I changed the buffer after o/n digestion to original elution buffer I had used but without GSH in it (i was replacing it with some other buffer earlier), and also I eliminated the AKTA purification step, and it seems i am much more happier.
Posted 26 September 2012 - 08:35 AM
"Thank you very much for contacting GE Healthcare with your question.
Unfortunately, your observation is not something easy to address—it needs more investigation.
Pretreatment of GSTrap column/glutathione sepharose resin does not help. The underlining issue comes from protein folding. GST-tag serves as a good carrier partner to keep fusion protein in solution. Yet, sometimes, the target portion of the fusion only stay soluble when interacting with the GST portion. And such interaction is so strong that even after proteolytic cleavage, thus two polypeptide chain, they still stay together in solution, as if cleavage did not occurred. I am not aware of much consensus currently to address this problem. One of my colleague worked around it by screen out a few different construct, i.e. GST fusion protein with slightly different linker sequences. The only way forward, with your current construct, is to test out different conditions and, hopefully, one combination can be found to warrant separation. Additives such as detergent, low concentration of chaotropic agents (urea) are the normal direction. I would not consider SEC (size exclusion chromatography), rather ion exchange in the presence of urea. Anionic exchange chromatography (AIEX), such as mono Q is most widely used. Yet, please understand that as long as the interaction between target and GST is not fully disrupted, the complex will behave as one species in solution, thus chromatography separation is not possible."