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Cleaved GST fusion protein retained on beads!


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9 replies to this topic

#1 mert1518

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Posted 03 March 2005 - 09:14 AM

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.

#2 tanyaaspinall

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Posted 04 March 2005 - 07:21 AM

:( 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!

#3 sharath

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Posted 06 March 2005 - 10:36 PM

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.
Sharath B.

#4 tanyaaspinall

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Posted 07 March 2005 - 03:25 AM

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!

#5 mert1518

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Posted 08 March 2005 - 09:50 AM

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?

#6 Esen

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Posted 26 January 2011 - 07:29 AM

6 years later I am having the same problem.. Could you please provide some updates as far as you can remember?
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
-Add prescission
-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

#7 vivekp

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Posted 12 February 2011 - 11:30 AM

add 0.1 % sarcosyl and then dialyze it against ur buffer..protein is in its native state...i have done it successfully

best
V

Edited by vivekp, 12 February 2011 - 11:32 AM.


#8 Esen

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Posted 24 February 2011 - 02:58 AM

Thanks vivek..

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.

#9 cdc2kinase

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Posted 26 September 2012 - 08:35 AM

I had a same problem and emailed GE customer center. Here is the reply I got (see below). Look like my protein is not stuck on the beads, it stuck on GST-tag and we can't do anything about it. I tried to elute with 4M UREA, but it was not successful. Only thing it worked partialy was eluting with 0.1% Sarcosyl like vivekp said.

"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."

#10 icemare

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Posted 10 June 2013 - 11:40 PM

Dear all,
I have completely the same problem although with resin and HaloTag fusion protein. After TEV cleavage practically all proteins retain on the beads.
I wonder if here is something more to say or if you could share which are the most efficient approaches to at least partly solve the problem.
Since I need fully functional protein I worry to use denaturants.
Thank you for any suggestions.




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