I have purified a recombinant protein and I am not getting any protein activity in activity assay. Protein contains histidine tag. Is there possibility that his tag is interfering with protein activity. Should I need to remove his tag?
Dear Anurag Dhiman
there is not a common rules it depends from the protein properties (eg. is your protein a metal binding protein?) and from the location of the tag (if you are unlucky and in the folding protein the tag is localized close to the active site and create some steric hindrance.
Of course if you have between protein and tag an enzyme cleavage site, the best way to answer to your question is, remove it by digestion and try again your enzymatic assay.
good luck
Manuele
Hello Anurag Dhiman,
the presence of a histidine tag can also interfere with the activity of your protein. It is an unusual event that occurs rarely. It depends on whether the tag is placed on N- or C-terminal of the protein. The addition of a histidine tag can also sometimes result in reducing the protein solubility. Also, in case your protein is a multimeric or oligomeric protein, the affinity tag may interfere with its structural conformation. Although this tag is more convenient due to its lower molecular weight, easy detection, and purification; one may make use of another tag such as GST or Maltose binding protein (MBP) that enhances protein solubility and stability.
Hi
We always run a simulation (MD) before we move to expression phase. Now its not too late, you can quickly do it and predict if your His-taq could interfere with your protein.
Good luck
If you suspect that the His-tag affects the activity of your target protein, especially if the tag is at the N-terminus, then remove it by digestion.
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