How does one go aboutHis-tagging - (Oct/02/2001 )

I have this protein that I'm going to clone into an expression vector such as pET. I am looking to over-express it and then purify it. To do this I have been told that His-tagging will allow me to do this effectively. I understand the concept that high-affinity columns can be used to purify the protien, but I don't understand how I am supposed to incorporate the poly-histidine tag into the protein?

Thanks

aoifa

The pET vector (there is a big choice) actually contains the His-Tag. All you have to do is clone your protein (coding sequence) into the vector. The vector also contains a start signal so any ATG at the beginning of your sequence is unnecessary. Check with Novagen Inc.