I want to design a primer to make a 2X Flag tagged protein.
The forward primer would have the sequence of 2xflag and the cds of the gene.
I also want to add a restriction site BglII (AGATCT) between the 2Xflag and CDS, so it would be convenient for sub-cloning of other flag tagged protein.
However, that mean the BglII site would be translated, and a Arg Ser linker would be formed between the tag and my target protein. I wonder whether this will affect the result? How people usually design the linker between tag and the target protein? Any specific criteria or recommended a.a. ,etc?
Thank you.
Hi,
in general terms having the Arg-Ser from the BglII site should not be a problem. It really depends upon what purpose you are expressing your protein(s) for, and the characteristics of your protein(s). If you intend to leave the 2xflag tags on (as you don't mention any protease sites) then it might even be beneficial as it can act as a short linker separating the tags from the protein. (If however your protein requires a native N-terminus then a C-terminal tag would be required.)
An alternative would be to use a BamHI site GGATCC which would give you a Gly-Ser enabling you to create a thrombin cleavage site Leu-Val-Pro-Arg/Gly-Ser. pET28 provides a good example of this.
Richard
Hi.
I think it should not be a problem. I expressed proteins with more amino acids between tag and protein of interest and it was working for my demand. If you want to get rid of your tag i would introduce a cleavage site for e.g. thrombin or TEV protease.
can you tell which terminal of your protein is going to be tagged, C or N terminal?
In terms of innocuous amino acids another alternative to BamHI (Gly-Ser) is ScaI (Ser-Thr).
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