epitope tagging of pcr products - (Oct/25/2009 )

hi
I am going express some intracellular proteins in mammalian cells and I want to to add an small epitope tag (myc, his. flag,...) to my reverese primer for detedtion of the expressed protein by ELISA or western blotting . I have some questions for this:
1. does adding small C terminal tags would interfere with activity of the protein (I search some commercial vectors in many of them tags are in N terminal).

2. is there any preferance in chosing a tag for c terminal use? I mean if some tags act better when add to c terminal.

3. is it necessary to put some spacer sequences between my protein and tag or I can add it directly to my protein, if yes what kind of spacer I can use.

many thanks

sara.r on Oct 25 2009, 06:57 PM said:

I don't think so, but you never can tell with proteins!

A bit of work has gone into this question. look at recent reviews about protein expression systems for high throughput structural genomics consortia for more details; I do know that GST tends to not work well at the C-terminus. We use GFP as a C-tag, with an N-terminal His tag, because NTA columns turn a lovely green as we load the lysate (this shows that both ends of the fusion are present, so the middle will be also!)

We have designed our system with TEV- and Precission-cleavable tags. This way the protease sites form an effective linker between the tag and the protein.
All the best with it.