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HaloTag Engineering for Enhanced Fluorogenicity and Kinetics with a Styrylpyridium Dye


Miró‐Vinyals, Carla; Stein, Alina; Fischer, Sandro; Ward, Thomas R; Deliz Liang, Alexandria (2021). HaloTag Engineering for Enhanced Fluorogenicity and Kinetics with a Styrylpyridium Dye. Chembiochem, 22(24):3398-3401.

Abstract

HaloTag is a small self-labeling protein that is frequently used for creating fluorescent reporters in living cells. The small-molecule dyes used with HaloTag are almost exclusively based on rhodamine scaffolds, which are often expensive and challenging to synthesize. Herein, we report the engineering of HaloTag for use with a chemically accessible, inexpensive fluorophore based on the dimethylamino-styrylpyridium dye. Through directed evolution, the maximum fluorogenicity and the apparent second-order bioconjugation rate constants could be improved up to 4-fold and 42-fold, respectively. One of the top variants, HT-SP5, enabled reliable imaging in mammalian cells, with a 113-fold fluorescence enhancement over the parent protein. Additionally, crystallographic characterization of selected mutants suggests the chemical origin of the fluorescent enhancement. The improved dye system offers a valuable tool for imaging and illustrates the viability of engineering self-labeling proteins for alternative fluorophores.

Abstract

HaloTag is a small self-labeling protein that is frequently used for creating fluorescent reporters in living cells. The small-molecule dyes used with HaloTag are almost exclusively based on rhodamine scaffolds, which are often expensive and challenging to synthesize. Herein, we report the engineering of HaloTag for use with a chemically accessible, inexpensive fluorophore based on the dimethylamino-styrylpyridium dye. Through directed evolution, the maximum fluorogenicity and the apparent second-order bioconjugation rate constants could be improved up to 4-fold and 42-fold, respectively. One of the top variants, HT-SP5, enabled reliable imaging in mammalian cells, with a 113-fold fluorescence enhancement over the parent protein. Additionally, crystallographic characterization of selected mutants suggests the chemical origin of the fluorescent enhancement. The improved dye system offers a valuable tool for imaging and illustrates the viability of engineering self-labeling proteins for alternative fluorophores.

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Additional indexing

Item Type:Journal Article, refereed, further contribution
Communities & Collections:07 Faculty of Science > Department of Chemistry
Dewey Decimal Classification:540 Chemistry
Scopus Subject Areas:Life Sciences > Biochemistry
Life Sciences > Molecular Medicine
Life Sciences > Molecular Biology
Physical Sciences > Organic Chemistry
Uncontrolled Keywords:Organic Chemistry, Molecular Biology, Molecular Medicine, Biochemistry
Language:English
Date:10 December 2021
Deposited On:09 Jan 2023 08:12
Last Modified:30 Jan 2024 02:37
Publisher:Wiley-VCH Verlag
ISSN:1439-4227
OA Status:Green
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1002/cbic.202100424
PubMed ID:34609782
Project Information:
  • : Funderarie Skłodowska-Curie
  • : Grant IDH2020-MSCA-IF-2017
  • : Project Title
  • Content: Published Version
  • Licence: Creative Commons: Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)