Question about Tag - (May/01/2006 )
I want to know that in pET32 a there is Trx-Tag, His-Tag, S-Tag, and then His-Tag, why there are so many tags and in at diffferent positions.
with best regards
Thioredoxin is not simply a tag, but rather a fused protein designed to facilitate expression of attached proteins through reduction of disulfide bonds, thus increasing solubility. I personally prefer Glutathione-S-Transferase for expression of soluble proteins. Thioredoxin has a c-terminal his-tag in this construct for visualization through western or affinity chromatography after cleavage with thrombin.
The thrombin site is present to allow the cleavage of thioredoxin (prior to purification) from the desired protein which one might wish to study.
The S-tag is present on the n-terminus and the His-tag is present on the c-terminus for verification and purification of the protein using Ni-NTA or Cobalt. Having two tags is much better than having merely one for verification purposes. Lots of things bind to Ni-NTA non-specifically, which is why the S-tag is present for further verification. The Enterokinase site is there for cleavage of the S-tag. Factor Xa can cut off the His tags.