How to insert a Tag to the middle of a protein? - (Dec/08/2004 )

Hi,

I am going to insert a tag(about 10AA) into the middle of a protein. Since connectting tag to the N or C-terminal were proved interfering the function of the protein. It is a protein about 400aa. I have no idea how I can do that. Who can give me some suggestion. As I know, PCR probably can do that, but I don't know how it work. Any suggestion will be appreciated.

Daniel from UNL

It is a very complex thing to explain... Well, PCR allows you to insert fragments into your DNA. Therefore you need primers which contain a part of your DNA, a part where a restriction site (rs) is encoded and some overhangs (to be sure that your rs works). If your DNA allready contains a rs, you only need to amplify your fragment with this rs. But caution: it is better that every end of you fragment has its own rs! Otherwise, it is possible that your fragment may recirculate and you are not able to ligate it... Also, your DNA must have - of course - same rs as your fragment...

Hope this will help a little bit...

I'm wondering if the tag disrupt the function of your protein at either N or C-terminal, do you expect it won't disrupt the function when it inserted in the middle?? Usually, one won't put a tag in the middle of a protein since (1) the chance of disrupting function will be much higher than putting at N or C-end; (2) overall structure will be highly likely to be disturbed...
(3) detection of the tag will be more difficult...

You need to be careful regarding where to put the tag if this is really necessary. Two approaches you can try: (1) PCR 2 fragments first (the N part with the tag and the C part), then ligate 2 together, be careful to be sure it is in-frame after ligation; (2) custom gene synthesis if money is not the problem. Our lab has good relation with Epoch Biolabs' service and they are one of the pioneers in this field, check thyem out (~$1.4-1.5 per bp).

Thank your advice. It had been proved that insertion of a sequence in the protein didn't affect the function. The position is in the loop of the membrane protein, it is easy to be detected by immunocytochemistry.
Thank you!