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GST system purification - Insoluble protein
Posted by: michellediniz (IP Hidden, New member, 4)
Date: May 17, 2005 02:27PM
Hi. I have tried to purify a bacterial protein by GST fusion system. In a first moment, the fusion protein was in the pos-lysate pellet, but we solve this problem changing the induction conditions and adding triton 1%, tween20 0,2% and glicerol 5%. However, after the cleavage with thrombin, the protein of interest was insoluble again, mixed with resin (after centrifugation). How can I solubilize it?
Re: GST system purification - Insoluble protein
Posted by: 5'-ATCG (IP Hidden, Unregistered user, )
Date: May 17, 2005 04:59PM
See if your proteins stick to GST or resin or self-aggregation first.
if stick to GST or resin, try to ajust ionic strength (pH), salt concentration....to break this interaction. if self-aggregation, try to add some reducing agents to solubilize.
Re: GST system purification - Insoluble protein
Posted by: michellediniz (IP Hidden, New member, 4)
Date: May 20, 2005 04:34PM
I already verify all this possibilities. Thanks.
Re: GST system purification - Insoluble protein
Posted by: michellediniz (IP Hidden, New member, 4)
Date: May 20, 2005 04:34PM
I already verify all these possibilities. Thanks.
monomer-dimer equilibrium
Posted by: pinnochio (IP Hidden, New member, 2)
Date: May 24, 2005 04:47PM
Hi I have a 12.7 KDa bacterial protein expressed as a C terminal GST fusion in E.Coli BL21 cells. This protein is an excellent expresser but crashes out if it is too concentrated. I use upto 1mM TCEP in my purification buffers and have tried adding 1mM DTT too. But I can still see a dimer band in the SDS-PAGE. This would not be so much of a problem since I'm doing gel filtration and colelcting only the monomer peaks. But MALLS data of the protein indicates that it exchanges between a monomeric and dimeric form at an intermediate rate. This is a problem because my goal is to determine the structure of this protein. Can anyone please tell me; 1. Ways to avoid the dimer formation in the first place. 2. How to push this monomer-dimer equilibrium that is going on with my protein in one direction or the other so I can determine the structure of either the monomer or the dimer. (preferably the monomeric form) Any other ideas? Thanks, Pinnochio.
Re: GST system purification - Insoluble protein
Posted by: ahujamunish (IP Hidden, New member, 2)
Date: June 30, 2005 09:02AM
My GST - fusion protein expressed as incluion bodies.
I solublize my protein in 1.5% of Sarcosyl but after extraction protein is not binding to sepharose 4B. Even After dialysis protein wont bind to column. Any Suggestion thanks Munish ahuja
Re: GST system purification - Insoluble protein
Posted by: ahujamunish (IP Hidden, New member, 2)
Date: June 30, 2005 09:29AM
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Re: GST system purification - Insoluble protein
Posted by: kamal (IP Hidden, Unregistered user, )
Date: June 30, 2005 09:36AM
I cant help you sorry
Re: monomer-dimer equilibrium
Posted by: elixir (IP Hidden, New member, 3)
Date: March 26, 2006 07:54PM
Hi,
Try to do 2 preps, one with the DTT and one without , practically these are for monomer and dimer respectively... Incase you are still getting dimer, increase the concentration of DTT so that you can get the monomer out, else try to mutate one of the cysteines in your protein and stop the dimer formation and see whether this mutant is functional, then you can proceed with your structural determination... Hope this helps..
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