Hi, may I know
Thank you for any assistance!
Dear Jia Hui Tee
there is no general role about how the presence of C- or N-terminal histidine tag can affect protein folding and stabilty since it may change a lot on the basis of expression host and protein properties (e.g his the protein bind a metal as cofactor and histidine tag can compete with the binding of this metal? His histidine tag spaced from the globular protein domain and do not affect the interaction of the protein with other proteins?
There is not a general answer to this question since it depends a lot from protein properties.
In general, what i observed in the past that if you are expressing a protein in the E.coli citoplasm in the most of cases the His-Tag is located at the N-term since in this case using enzimes as TEV. or Fatt.Xa it can be removed almost completelly after protein purification.
On the contrary when the protein is expressed in hosts as pichia, mammalian (eg expi293 or CHO) or even periplams in E.coli, the tag is more frequently located at the C.-terms probably because the locations at N-term close to the leader sequence and it cleavage site, in some cases may affect in some cases the secretion process.
But i do not think that there is a general role.
good luck
Manuele
Also, as you already said aplphaFold can help you with that. Just go to the alphaFold3 server, make a few predictions with the tags on different termini. It is super easy to use. If it doesnt affect the structure, the common tags are mostly not affecting stability..
Combination of different tags can improve purity but I rarely use it, rather combine IMAC with SEC or ion exchange.
Good luck
Tim
I agree with Manuele. I can add that for proteins produced in bacteria, a C-term tag allows to purify only the fully translated products. For large proteins this can be important since stop of translation is a common problem (especially if the DNA has not been optimized for the codon usage in coli). Saying that if a small proportion of the protein is fully translated, the purification will be proportional. As Manuele said, in other host the N-terminal (or the C-terminal) can be modified or important for secretion, therefore the position of the tag should be considered accordingly.
Double tags allow to use sequential purification of the protein that will increase the purity of the protein. In my hands, 6 (or 10) His tag works fine (i.e. purification is good) if the protein is well expressed (and soluble). If protein expression level is low then more contaminants are co-purified. In this case a double tag may help.
Hope this is helpful. Best.
M
To predict protein structure, use tools like AlphaFold or Rosetta. For choosing N or C terminal tagging, consider the protein's function, localization signals, and structural stability, ensuring the tag does not interfere with active sites or folding. Experiment with both tags to determine the optimal position.
Hey there,
I agree with the previous answers on how to determine how the tag location may influence your protein. Since the effect of the tag very much depends on the properties of the protein, I would suggest making a few predictions with Alphafold like Tim mentioned, but at the end the only way you can be sure on how the tagged protein behaves will be by expressing it and trying it out. If in your Alphafold predictions you see some issues, I suggest putting a linker between your target protein and the tag to check if it improves tag accessibility etc.
For tags, I would recommend using a Strep-tagII or Twin-Strep-tag rather than a His-tag. The Strep-tag usually does not interfere with protein structure and function, and factors like competing with metal cofactors of the proteins are not a concern with Strep-tag. In mammalia cell cultures, proteins native to the host that have histidine residues can often show up as impurities in His-purification, but this is not an issue with Strep-tag purification. So if you would prefer having one rather than two tags, I would always recommend the Strep-tag for a higher purity and yield, especially when working in mammalia, yeast or insect cells.
Good luck!
Christel
Besides structure prediction, you can always run a simple alignment of your protein with related proteins from other species. Frequently, you will find one end of the protein to be more conserved than the other. As conservation (in length or sequence) always indicates functional importance, choose the less conserved end for adding the tag.
Beware that the situation is more complicated with proteins that have targeting signals (such as cleavable or non-cleavable signal peptides). These may not be conserved but of course are relevant for function.
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