I want to clone a gene in a vector with His-tag. My protein is an enzyme and its active site is in the center. Is there any difference between adding an His-tag at the C-er or N-ter of the protein?
The short answer is: Do both and try which works better.
It totally depends on your protein, which end is "loose" and exposed on the surface of the protein, which is necessary for binding to the metal-loaded column. If a structure of your protein is available you can also predict which end of the protein is more promising for a tag. If both ends are burried a poly-Gly linker can help between the tag and the protein of interest.
There's plenty of things one can argue for here. Some of the common ones are that many wish to cleave the tag off after purification with TEV or 3C protease. For this, an N-terminal tag is better as it doesn't leave too much extra in the protein after cleavage (if you cleave a protein with TEV protease, you will end up with a single G or S residue in the N-terminus, whereas if the cleavage site was at the C-terminus you would have several extra amino acids).
On the other hand, some people like to put the tag on the C-terminus to prevent C-terminal degradation. It's good to try both separately if you are working with a new protein construct. Actually, some choose to have both termini tagged in a single protein to increase affinity for the Ni-NTA matrix.
Also, consider trying other tags (maltose binding protein, glutathione S-transferase, thioredoxin, GFP etc.) if you run into solubility problems or end up with a lot of unspecific binding in Ni-NTA. GFP should also help you visualize and quantify expression, although this is more often used with membrane proteins.
Hope this helps,
-Arne
Normally does not same, IF your protein has the Ct exposed to the solvent, the advantage of cloning the his-Tag in the Ct is that warrant that all protein that will purify subsequently are full length
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