Protein tags are short sequences of amino acids that are genetically fused to target proteins to aid in their purification, facilitate their detection or study their function. Despite their versatility, these tags may interfere with normal protein expression and/or function, complicating the interpretation of experimental data. A new study using genetically engineered mice with tagged proteins demonstrates that the effects of tags on protein expression depend on cell and tissue contexts. The study also characterizes two mechanisms by which terminal tag fusions can cause unwanted tissue-specific changes in protein expression.

In this study, Taylor et al. used mouse models that had been genetically modified to express two tags—the auxin-inducible degron (AID) and the fluorescent protein Clover—at the C terminus of NCAPH2, a protein involved in mitotic chromosome assembly and segregation. AID is a useful technique to rapidly and reversibly deplete proteins of interest in cellular and animal systems. Depletion is achieved by exposing cells or animals to auxin, a ligand that allows auxin-binding receptor TIR 1 (which also needs to be exogenously expressed in the transgenic organism or cell line) to target the AID‐tagged protein for degradation by the proteasome.

To investigate the effect of the C-terminal AID:Clover tags on NCAPH2 expression, the team performed western blots on tissue lysates from heterozygous mice (Ncaph2AID:Clover/+; Rosa26Tir1/Tir1) and quantified the expression of tagged and wildtype NCAPH2. They found that the ratio of tagged to wildtype NCAPH2 varied greatly across tissues, with the tagged protein being expressed at higher levels than the untagged protein in the brain and at lower levels in the small intestine.

Further experiments revealed that in the small intestine, the reduced expression of tagged protein was caused by leaky degradation in the AID system, specifically in this tissue. By contrast, in the brain, a tissue with a high proportion of post-mitotic cells, the increased expression of tagged protein was caused by the inactivation of endogenous degrons by the tag fusion. These new insights could help design better tagging strategies that prevent tag-dependent experimental artifacts.

Original reference: Taylor, G.C.A. et al. PLoS Genet. 21, e1011830 (2025)