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I'm thinking about the 3-dimentional structure of fusion protein (recombinant protein). If my target protein has a GST tag(Glutathione - S- Transferase), does this target protein has its own structure as native structure or the GST tag makes the fusion protein another new structure ?
In order to know that with any kind of certainty the structure of the protein has to be determined twice: once with and once without the GST tag--which has been done for at least one case. In that case both tagged and untagged protein structures were the same and the conclusion was that GST does not induce an alternate fold to the tagged protein--nor did the fused protein affect the structure of the GST "domain" of the fused protein. Once this was shown for one or possibly two cases, it is now assumed that the two "domains" of the fusion protein fold independently and, while GST may help an otherwise denatured and insoluble protein to fold, the fold you get is the same as the native fold of the protein. It is difficult to impossible to get fusion proteins to crystallize, so in general you can't "prove" that the fold you see (after removing the GST tag) is native, but that will be the assumption.
You could try it. I suspect gel electrophoresis wouldn't tell you very much. SDS-PAGE effectively denatures the proteins and you would only see significant changes in MW--something you expect to see in comparing the fusion product to either GST or protein alone anyway. Native gels might show structural differences, but what would you compare the fusion protein to? On a gel, I mean. You could also look at ORD/CD spectra, but that really only tells you something about the composition of helices or strands and gives some sense about whether the protein is folded or denatured. You might try chemical modifications. There again I have trouble imagining what you would compare to what to get a meaningful answer. For my money, direct structural comparison is the "best" way to answer the question, but it is certainly worth thinking about alternatives.
If the fused protein is an enzyme, you could look for changes in activity--either in the fusion product and/or after removing the GST (or whatever) tag. That's assuming you have a source of known-to-be-native protein to compare the activity against. This would be a phenomonological comparison. You'd still have to explain how it relates to putative differences in structure induced by the GST.
Oh, that means they alrealdy have some experiments for this, right ? Could you please give me detailed information involving in this problem ?
Also, I used to work with GST fusion protein for a while(expression, purification and also mice immunization using the fusion protein - my protein has the antigenic characteristic), it seems that SDS-PAGE cannot tell the difference in the structure with and without GST.
Although we attained good result on mice immunization, it cannot conclude that my protein remains all the 3-D structure (only part(s) in the protein takes part in the antigenic role).
Is there any posibility for the hypothesis that the enzyme still remains its 3-D structure, but the enzyme activity decrease because of GST "domain" (if have) ?
Hmm. Well, now having said all that, I can't put my finger on the reference(s) I was thinking of. The one I remember best probably isn't the world's best paper and didn't prove the point for the structure of an entire protein fused to GST. There was a paper published maybe more than 10 years ago that showed that a peptide derived from a protein whose crystal structure was known (I don't remember now which protein it was) adopted a structure that was the same in both the fusion protein and the native structure. The question was, for a small peptide--something considerably smaller than the carrier protein, GST--would the larger protein induce a different stucture on such a peptide, or would the peptide retain its native conformation, and the answer, at least for this peptide, was that it retained its native conformation. I'll keep looking for the specific reference(s) and if I find it/them I'll post them here.
I wouldn't expect to see any difference in "structure" on SDS-PAGE, only differences in MW and if the peptide is fairly small, you won't see much difference in MW between the fused and unfused GST.
That antibodies raised against peptides fused with GST recognize native protein is pretty good evidence that the peptides fused with GST adopt native conformations. I don't necessarily consider it "proof," but it is a reasonalbe assumption. I can also envision situtations where the presence of GST could affect the activity of a fused enzyme even though the enzyme is also in its native conformation. Access to the active site could be mechanically restricted if the GST is physically in the way, for example. You might expect the increase in mass of the fused enzyme to have a small effect on the rate constants--although I would expect mass effects to apply more to small MW substrates than enzyme.
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