Molecular cloning using an ezh2 insert - (Nov/27/2014 )

Hi! :)

I am confused about his-tags and the way they work. If i am cloning the EZH2 gene in a vector/plasmid, why does the his tag have to be sited on the amino terminal of the ezh2 protein?

Thank you!

Basically there are two reasons why you might need to tag at the N-terminus...and one of those isn't really a reason, just a convenience:

One scenario is that tagging at the C-terminus causes the protein to misbehave in some fashion (doesn't transport properly, doesn't fold properly, loses some interaction, etc.), this is very valid reason for concern and definitely a case where you would avoid the C-terminus. The other scenario is that the plasmid you are inserting it into is a N-terminal tagging plasmid -the tag is already in the plasmid and upstream of the multiple cloning site, so you don't have to amplify the insert with a His tag in your primers.

bob1 on Thu Nov 27 17:33:43 2014 said:

Basically there are two reasons why you might need to tag at the N-terminus...and one of those isn't really a reason, just a convenience:

 

One scenario is that tagging at the C-terminus causes the protein to misbehave in some fashion (doesn't transport properly, doesn't fold properly, loses some interaction, etc.), this is very valid reason for concern and definitely a case where you would avoid the C-terminus. The other scenario is that the plasmid you are inserting it into is a N-terminal tagging plasmid -the tag is already in the plasmid and upstream of the multiple cloning site, so you don't have to amplify the insert with a His tag in your primers.

Perfect! Thank you so much!