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preference of N/C terminus for tagging - (Apr/23/2010 )

Hi,

Does anyone know of bacterial proteins where tagging at the N instead of C terminus (or viceversa) has had major consequences on protein folding?Do proteins prefer to be tagged at one particular end over the other and is there some bioinformatics tool that can predict the influence of a tag on protein structure? i have been searching out stuff on similar lines but it has been hard to get a clear answer.

Help is much appreciated :wacko:

Thanks
Deedee.

-DeeDee-

DeeDee on Apr 24 2010, 07:42 AM said:

Hi,

Does anyone know of bacterial proteins where tagging at the N instead of C terminus (or viceversa) has had major consequences on protein folding?Do proteins prefer to be tagged at one particular end over the other and is there some bioinformatics tool that can predict the influence of a tag on protein structure? i have been searching out stuff on similar lines but it has been hard to get a clear answer.

Help is much appreciated :)

Thanks
Deedee.

It is difficult to predict the effect of tagging a protein. It can totally destroy biological activity and/or lower (or abolish) expression levels. I am not aware of any bioinfo tool, however common sense often helps. For example, if your protein needs a free N-terminus for activity it is not a good idea to place a tag there! For example, with recombinant antibodies (eg scfv), conventional tags are always placed at the C-terminus. N-terminal tagging usually destroys antibody binding activity although some labs have recently shown that very small N-tags (4 aa) can be tolerated.

You would also not want to use an N-terminal tag on a secreted protein.

Hope this helps

-klinmed-

The problem with this protein I'm working on is that both the N and the c terminal domain movement is required for the active site lying between them... so there is no obvious way to predict which end is more crucial for movement...I was therefore wondering what criteria people follow before tagging (if at all there is a conscious decision making involved here) or what patterns emerge from general observation of tagged proteins.For example: if the N-terminus is contains small amino acids like glycine and proline that are known to bend protein shape(please correct m if I'm wrong!) then would you avoid tagging there... or is the end that is farthest away from the active site better preferred? These are rather vague questions but I'm new to this entire thing and dont know what exactly to think:)..... I use a His-tag,would a change of tag like GST help better in cases where histag dosent work??

-DeeDee-