The tags are usually used to detect your protein of interest with an antibody. You want to allow enough space for your antibody to bind to the tag without steric hinderance. In western blotting, where your protein is denatured, a linker is probably not needed. For recognition of the folded protein, e.g. in Immunoprecipitation, FACS sorting etc. you want to be on the safe side and add a highly flexible (Gly-Ser) linker. How much of a linker is needed between multiple copies of the same tag added for increased sensitivity depends on the specific tag-antibody pair.

Annemarie, I appreciate your answer. I got it.

GS linkers are not good recognition sites for proteases, where we have observed proteolysis in linker-connected constructs, the cleavage occurred outside the actual linker. In constructs designed for potential therapeutic use, such as antibody single-chain constructs, they are frequently preferred because of their low immunogenicity. Less repetitive linkers are needed for in-vitro evolution strategies involving gene shuffling, as the limited sequence diversity of a Gly-Ser linker may result in changes in the linker length (https://www.bioc.uzh.ch/plueckthun/pdf/APpub0135.pdf). Rigid linkers are definitely preferable when generating constructs for crystallography, as in this context you want to minimize conformational heterogeneity. Also, rigid linkers can be used to bring two domains into a defined relative orientation ideal for a specific purpose (https://www.bioc.uzh.ch/plueckthun/pdf/APpub0409.pdf, https://www.bioc.uzh.ch/plueckthun/pdf/APpub0408.pdf). In eukaryotic expression, flexible tags, especially at the C-terminus, may serve as signals for proteosomal degradation.

Whether to best use a GS linker, a flexible linker with a different sequence or a rigid linker depends very much on the specific application and on how much is already known about the structure and spacial requirements of the domains to be linked. In the specific case described in the question, to space out multiple flexible epitopes to allow multiple antibodies to bind simultaneously to the same construct, with the purpose to get better detection sensitivity, maximal flexibility seems to be of foremost importance to avoid sterical clashes between the multiple large antibody molecules trying to bind to the tag.

Annemarie Honegger Dear Annemarie,

Do you have the reference to this interesting point? "In eukaryotic expression, flexible tags, especially at the C-terminus, may serve as signals for proteosomal degradation."

Search for "Degron"

e.g. Article N-degron and C-degron pathways of protein degradation

Article Tying up loose ends: The N-degron and C-degron pathways of p...

Annemarie Honegger Thank you very much!