I would like to know suggestions for a detection and purification tag for a 150 kDa recombinant protein that is to be expressed in insect cells. This protein would be used later for immunization experiments in animals.
What would be the right place : N or C terminal fusion? and do i have to include more than 1 tag?
I would prefer to use a tag that doesn't affect the property of this protein and has lesser steps in downstream purification process and preferably less or non immunogenic tag
Thanking you
Use a cleavable tag, such as one with a TEV or thrombin cleavage site. That way, after you use the tag to purify the protein, you can remove it. It can go at either the N- or C-terminus. It's probably a good idea to make both constructs, since you can't always predict which will work better.
Do you have any posttranslational processing, requirement for and subsequent removal of signal sequences etc. ?
Do you have any information on the structure of your protein and if a terminus is buried (i.e. adding a tag might affect proper folding and function)?
If in doubt, I'd add it at the C-terminal.
If you can get an antibody against your protein, you could make an affinity column and omit the tag completely :)
You can try to purify protein with HIS tag. In general, labels have little effect on experiments. It can be placed on the N-terminal or C-terminal. The HIS tag is relatively small and generally does not affect the properties of the protein. If you are still worried about the impact of the tag, you can arrange affinity purification and HIS tag back screening after subsequent immunization.
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