What's good and bad about C-tem and N-term His tag? - (Apr/22/2007 )
I'm studying the function of a protein. I'm thinking of inserting the corresponding cDNA sequence into a vector and transformed into E.coli to get some purified recombinant protein. Got 2 questions:
1. I'd like to add a His tag for purification and immunostaining purpose. Does it matter whether the His-tag is C-terminus or N-terminus? Why somebody use C-term His while others use N-term His? What's the good and bad aspect of each one?
2. Does the additional His tag gonna affect the protein function in any way? What experiment can I do to check whether the protein function is affected or not?
Hello Ohmyhead,
it depends on your protein where to add the His-tag.
When you have a signal sequence at one end of protein the His-tag might disturb the interaction of this end with its partner. This can be due to the positve charge of the histidines that can block needed negative charges - on one hand with the interacting partner on the other hand with your protein itself.
So when you already know that there is an important sequence at one end of your protein you should choose the other one to add you tag.
It's difficult to say if there is a negative influence of the tag anyway. It depends on the protein after all.
To see if your protein function is affected you could try different tags or try both C-terminus or N-terminus in comparison.
Hope this helps you to decide... 
Greetings,
Chakchel
1. I'd like to add a His tag for purification and immunostaining purpose. Does it matter whether the His-tag is C-terminus or N-terminus? Why somebody use C-term His while others use N-term His? What's the good and bad aspect of each one?
2. Does the additional His tag gonna affect the protein function in any way? What experiment can I do to check whether the protein function is affected or not?
best is to check both, C- and N-tagged, to demonstrate that the tag does not affect the function;
membrane receptors with extracellular domains are predominantly N-terminal; tagging the N-terminus gives the opportunity to target the His-tag without lysing the cell
1. I'd like to add a His tag for purification and immunostaining purpose. Does it matter whether the His-tag is C-terminus or N-terminus? Why somebody use C-term His while others use N-term His? What's the good and bad aspect of each one?
2. Does the additional His tag gonna affect the protein function in any way? What experiment can I do to check whether the protein function is affected or not?
best is to check both, C- and N-tagged, to demonstrate that the tag does not affect the function;
membrane receptors with extracellular domains are predominantly N-terminal; tagging the N-terminus gives the opportunity to target the His-tag without lysing the cell
Thanks guys, all useful suggestions. My protein is a secretion protein. It is made intracellular and then secreted into culture media so I think I need to try both C- and N-tagged.