# EpicTope: predicting and validating non-disruptive epitope tagging sites

## TL;DR

EpicTope is a tool that predicts where to insert tags in proteins without disrupting their function, validated in zebrafish.

## Contribution

EpicTope introduces a novel scoring function combining structural and functional features to predict non-disruptive epitope tagging sites.

## Key findings

- EpicTope predicted internal Smad5 tags rescued zebrafish mutant embryos, unlike N- and C-terminal tags.
- Optimal internal and C-terminal Hdac1 tags rescued hdac1 mutant embryos, while N-terminal tags did not.
- Functionally tagged Smad5 and Hdac1 proteins were accessible to antibodies in various assays.

## Abstract

Epitope tagging is a valuable technique enabling the in vivo identification, tracking and purification of proteins. We developed a tool, EpicTope, to facilitate this method by identifying amino acid positions most suitable for epitope insertion. Our method uses a scoring function that considers protein sequence secondary and tertiary structural features, solvent accessibility, and disordered binding regions to determine locations least disruptive to the protein function. We validated our approach on the zebrafish Smad5 and Hdac1 proteins. We show that multiple predicted internally tagged Smad5 proteins rescue zebrafish smad5 mutant embryos, while the N- and C-terminal-tagged variants do not, as predicted. Similarly, we found that optimally predicted internal and C-terminal Hdac1 tags rescued hdac1 mutant embryos, while a less-optimal N-terminal tag did not. We further show that these functionally tagged Smad5 and Hdac1 proteins are accessible to antibodies in whole-mount zebrafish embryo immunofluorescence, by western blotting and by immunoprecipitation from embryo extracts. Our work demonstrates that EpicTope is an accessible and effective tool for designing epitope tag insertion sites.

Summary: The EpicTope computational program predicts optimal insertion sites for generating epitope-tagged proteins, validated in zebrafish by seven V5-tagged sites in Smad5 and Hdac1, and tested by functional assays of mutant phenotypic rescue, immunofluorescence, immunoprecipitation and western blot analysis.