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I am attempting to purify a protein complex of two proteins. The Protein "A" and Protein "B" have been co-expressed in media containing N15-Cys. Protein "A" has a 6-histadine tag. I am able to purify this complex via a Ni-NTA column with good results. I now have the Protein "A-labled/B-labled" complex with some excess Protein "A." The "A/B" complex has a molecular weight of ~60 kDa.
I want to exchange the labeled Protein "B" in the "A/B" complex for un-labled Protein "B."
I have also expressed GST-tagged Protien "B" in media without N15-Cys. With GST-tagged Protien "B" (unlabled) bound to a GSH column, I applied the "A-labled/B-labled" complex to the GSH column. However, almost all of the protein remained on the column after on column thrombin cleavage, 1 hour at room temperature (thrombin cleavage site at C-term of GST). This was my preferred method since it would elimnate the GST.
If I use glutathione elution on the GSH column, then I will have to do thrombin cleavage in solution, which will yield more efficient cleavage. I will then have a mixture of my target Protein "A-labled/B-un-labled," monomeric GST, and dimeric GST. I can separate monomeric GST from "A-labled/B-un-labled" by size exclusion chromotography.
Here is my question:
How do I elimnate the GST dimer when the molecular weight is so close to the "A-labled/B-un-labled" dimer? (GST ~52 kDa, "A-labled/B-un-labled" ~60 kDa)
Caveats:
We do not have monomer only GST.
The pI's are too close to use ion-exchange.
Protein "A" must be co-expressed as it does not express well alone.
Many Thanks,
Sarah Presley
Ph.D. Candidate
University of Tennessee
Department of Biochemistry, Molecular, and Cellular Biology