Abstract
While protein tags are ubiquitously utilized in molecular biology, they harbor the potential to interfere with functional traits of their fusion counterparts. Systematic evaluation of the effect of protein tags on function would promote accurate use of tags in experimental setups. Here we examine the effect of green fluorescent protein tagging at either the N or C terminus of budding yeast proteins on subcellular localization and functionality. We use a competition-based approach to decipher the relative fitness of two strains tagged on the same protein but on opposite termini and from that infer the correct, physiological localization for each protein and the optimal position for tagging. Our study provides a first of a kind systematic assessment of the effect of tags on the functionality of proteins and provides a step toward broad investigation of protein fusion libraries.
Highlights
Report Protein tags are essential for a variety of assays in biology - from affinity tags for protein purification to fluorescence tags for visualization
We use a Green Fluorescent Protein (GFP) tag as a test case and rely on a recent comparison made between two whole-genome libraries of strains, each encoding one protein fused to GFP at either the N’[5] or C’[6,7].In this comparison it was shown that 515 proteins in yeast are differentially localized when tagged in the opposing termini (Fig. 1A)
While protein function can be impaired without displaying a mis-localization, it is clear that a difference in localization affects the capacity of a protein to function properly in a cellular context
Summary
We chose these proteins to test our method: which tagged terminus represents the physiologically relevant localization of these proteins? To systematically address whether an N’ or C’ tag better represents the correct cellular localization of a given protein, we established a pairwise competition approach that relies on the assumption that there would be a growth advantage to the strain carrying the correctly localized protein form (Fig. 1B).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
