GST-fusion won't elute! - (Dec/08/2007 )

Hi

I am trying to purify a GST-fusion protein but am having difficulty eluting it from the glutathione sepharose (Amersham Biosciences). The product manual says I can go up to 50 mM glutathione and 500 mM NaCl however even in these conditions 80% of my protein remains on the beads.

Any suggestions? I don't want to denature the protein as I need it to be functional for downstream applications,

Thanks in advance!

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I'd try even higher levels of glutathione, even up to a couple of 100 mM. You can always remove it later.

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a guy in my lab had problems with it. the protein he was working on turned out to be completely insoluble, due to many cistine residues.
so, perhaps take a good look at the protein, and look out for "things" that might make the protein a little difficult to work with.

V

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If that's the case, I suppose you could try denaturing the whole thing, eluting and then trying to refold. You might need to consider on-column digestion, and then purification by ion-exchange or gel filtration.

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Depend on the downstream applications and the quality of bound protein, there may not be necessary to elute the protein out.

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I purified my GST-fusion protein with 50mM Tris.Cl/5mM reduced glutathione pH8.0. From memory (it was a while ago), it was best if the RG was made fresh.
Not sure if this helps

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Thanks everyone,
I will try even higher concs of glutathione first, and yes I do need to get it off the beads as I need to purify it further by IEC.

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Frangioni and Neel (Anal Biochem 1993, 210: 179-187) describe the addition of 0.1 % Triton X-100 or 2 % N-octylglucoside to improve elution, of course the presence of detergents may be harmful for your downstream applications, but maybe it's worth a try?

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Thanks dpo!!
Very useful paper - I can always get rid of the detergents by dialysis later
P

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