Before removing the GST-Tag my enzyme produce cysteine but after removing the tag it is not showing its activity.
Plant Molecular Biology, Proteomics
Hi Rashid,
it might be that the removal of the GST-tag during the purification process reduces the solubility of your protein of interest, which might lead to decreased activity. To check on this, you can combine your activity assay with SDS-PAGE comparing samples from every step of the purification process, especially before and after removing the tag.
An other reason could be a buffer change during purification or tag removal not beneficial to your enzyme.
Best wishes Thomas
Hi there,
GST tag may act as a chaperone : solubilizing and stabilizing fusion partner structural integrity. The removal of the tag might have promoted partner denaturation/aggregation/precipitation and therefore enzyme inactivation.
Hi Rashid,
it might be that the removal of the GST-tag during the purification process reduces the solubility of your protein of interest, which might lead to decreased activity. To check on this, you can combine your activity assay with SDS-PAGE comparing samples from every step of the purification process, especially before and after removing the tag.
An other reason could be a buffer change during purification or tag removal not beneficial to your enzyme.
Best wishes Thomas
So Mr Downvoting et al. is back! Any explanation (I mean at science level) to justify such a vote???? The stabilizing effect of GST or MBP in fusion protein has been known for years already. Any breaking news I would have missed about this?
@Dominique
No idea. At a guess, I would say that wouldn't work if expressing in prokaryotes. *shrugs*
I agree completely with Dominique and Thomas. In our hands GST and MBP are two of the worst fusions for giving you falsely soluble protein, i.e. the fusion is solubilizing/stabilizing your protein but has not helped it in terms of correct folding. As soon as you remove these you lose protein stability/function and frequently see precipitation.
A good test of stability (if it isn't already precipitating) would be SEC of your cleaved protein-if it is monodispersed then it is probably folded OK and not aggregating so the activity problem may be inhibition by a buffer component e.g. EDTA for cation dependent proteins, reducing agents or detergents.
For us the best fusions to help folding, and therefore long-term stability even after cleavage, are SUMO, Trx and perhaps Z-tag.
P.S. downvoters if you have something to say then add to the debate instead of anonymously putting down someone else's carefully thought out contribution to a thread! It is really annoying to give something your best shot and be downvoted and it doesn't motivate people to help others out in forums like this.
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