A non-fluorescent HaloTag blocker for improved measurement and visualization of protein synthesis in living cells.

Laurie D Cohen,Ayub Boulos,Noam E Ziv

doi:10.12688/f1000research.23289.1

Abstract

Background: HaloTag is a modified bacterial enzyme that binds rapidly and irreversibly to an array of synthetic ligands, including chemical dyes. When expressed in live cells in conjunction with a protein of interest, HaloTag can be used to study protein trafficking, synthesis, and degradation. For instance, sequential HaloTag labeling with spectrally separable dyes can be used to separate preexisting protein pools from proteins newly synthesized following experimental manipulations or the passage of time. Unfortunately, incomplete labeling by the first dye, or labeling by residual, trapped dye pools can confound interpretation. Methods: Labeling specificity of newly synthesized proteins could be improved by blocking residual binding sites. To that end, we synthesized a non-fluorescent, cell permeable blocker (1-chloro-6-(2-propoxyethoxy)hexane; CPXH), essentially the HaloTag ligand backbone without the reactive amine used to attach fluorescent groups. Results: High-content imaging was used to quantify the ability of CPXH to block HaloTag ligand binding in live HEK cells expressing a fusion protein of mTurquoise2 and HaloTag. Full saturation was observed at CPXH concentrations of 5-10 µM at 30 min. No overt effects on cell viability were observed at any concentration or treatment duration. The ability of CPXH to improve the reliability of newly synthesized protein detection was then demonstrated in live cortical neurons expressing the mTurquoise2-HaloTag fusion protein, in both single and dual labeling time lapse experiments. Practically no labeling was observed after blocking HaloTag binding sites with CPXH when protein synthesis was suppressed with cycloheximide, confirming the identification of newly synthesized protein copies as such, while providing estimates of protein synthesis suppression in these experiments. Conclusions: CPXH is a reliable (and inexpensive) non-fluorescent ligand for improving assessment of protein-of-interest metabolism in live cells using HaloTag technology.

Highlights

The 33 kDa HaloTag protein[1] is a modified haloalkane dehalogenase enzyme which bonds covalently to a variety of synthetic ligands, including chemical dyes
We reasoned that reporter proteins containing one HaloTag binding site and one copy of a fluorescent protein (FP) would be useful in this regard, as this would allow normalization of HaloTag ligand fluorescence to FP fluorescence and calculation of fractional HaloTag labeling values, independent of fusion protein expression levels
The findings described above suggest that CPXH is a potent non-fluorescent, cell-permeable, and non-toxic HaloTag ligand

Summary

Introduction

The 33 kDa HaloTag protein[1] is a modified haloalkane dehalogenase enzyme which bonds covalently to a variety of synthetic ligands (reporters), including chemical dyes. Methods: Labeling specificity of newly synthesized proteins could be improved by blocking residual binding sites. Results: High-content imaging was used to quantify the ability of CPXH to block HaloTag ligand binding in live HEK cells expressing a fusion protein of mTurquoise[2] and HaloTag. Full saturation was observed at CPXH concentrations of 5-10 μM at 30 min. The ability of CPXH to improve the reliability of newly synthesized protein detection was demonstrated in live cortical neurons expressing the mTurquoise2-HaloTag fusion protein, in both single and dual labeling time lapse experiments. No labeling was observed after blocking HaloTag binding sites with CPXH when protein synthesis was suppressed with cycloheximide, confirming the identification of newly synthesized protein copies as such, while providing estimates of protein synthesis suppression in these experiments. Conclusions: CPXH is a reliable (and inexpensive) non-fluorescent ligand for improving assessment of protein-of-interest metabolism in version 2 (revision)

Methods

Results

Conclusion