His tag introduction in to gene and primer design - (Mar/11/2013 )
I want to introduce his taq into my protein and I need following information (Currently i am using pET26b vector)
1. If i remove the termination codon of the gene during designing the primer and introduce restriction site results incorporation of 5-10 amino acids before the his taq than wat could be the possible effect of these sequences on protein?
2. If i want to remove his tag after purification of protein of interest how should i design the reverse primer?
3. If i want to introduce the pelB sequence of expression vector, is it necessary to remove the initiation codon from the start of gene durig primer design?
I will answer #2. If you want to remove the his tag after purification you need to have a cleavage sequence following the His tag. This can be engineered into the primer. Look up an enterokinase cleavage sequence. It is used in the pET32 vectors and you can just use it. Then you will need a enterokinase cleavage/capture kit for the removal of the tag.
#1. I am not sure what you are trying to accomplish here.
The overall structure of your primers should be something like this
Forward Primer: 5' Restriction site----Start Codon----His6 Tag-----Enterokinase Site----Gene Start 3'
Reverse Primer 5' Restriction site----Stop Codon-----Gene end 3'
You might be overthinking this a little bit. The forward primer will be long (80 bases or so) but don't worry the PCR should still work.