Internal epitope tagging informed by relative lack of sequence conservation.

Leonard Burg,Tristan Bonawitz,Richard Waring,Karen Zhang,Viktorija Grajevskaja,Darius Balciunas,Gianfranco Bellipanni

doi:10.1038/srep36986

Abstract

Many experimental techniques rely on specific recognition and stringent binding of proteins by antibodies. This can readily be achieved by introducing an epitope tag. We employed an approach that uses a relative lack of evolutionary conservation to inform epitope tag site selection, followed by integration of the tag-coding sequence into the endogenous locus in zebrafish. We demonstrate that an internal epitope tag is accessible for antibody binding, and that tagged proteins retain wild type function.

Highlights

High-throughput experimental approaches such as chromatin immunoprecipitation and immunoprecipitation mass spectrometry rely on strong and highly specific recognition of the protein of interest using antibodies[1,2]
We hypothesized that evolutionary conservation can be used to guide internal epitope-tagging approaches
We posit that regions of relatively low conservation are unlikely to be involved in the critical function of the protein

Summary

Introduction

High-throughput experimental approaches such as chromatin immunoprecipitation and immunoprecipitation mass spectrometry rely on strong and highly specific recognition of the protein of interest using antibodies[1,2]. The second method is to express the protein of interest with a well-characterized epitope such as V5, FLAG or Myc[3] This enables researchers to use commercially available, validated reagents and protocols for downstream applications. It is rarely possible to test if the tagged protein matches the functionality of the wild type counterpart at normal cellular concentration The limitations of these traditional strategies can be overcome by inserting an epitope-coding sequence into the endogenous locus as is frequently done in yeast[4]. Internal integration of the epitope tag offers an important advantage: frameshift mutations can be isolated in parallel with epitope tagging Together, these can be used to characterize loss-of function phenotype(s) and to determine if the epitope-tagged protein is functionally wild type. We hypothesized that protein segments of variable length, in addition to relatively low conservation, would be the most suitable targets for internal epitope tagging

Methods

Results

Conclusion