Split-HaloTag imaging assay for sophisticated microscopy of protein–protein interactions in planta

Meinen, Rieke; Weber, Jan-Niklas; Albrecht, Andreas; Matis, Rainer; Behnecke, Maria; Tietge, Cindy; Frank, Stefan; Schulze, Jutta; Buschmann, Henrik; Walla, Peter Jomo; Mendel, Ralf‐R.; Hänsch, Robert; Kaufholdt, David

doi:10.1016/j.xplc.2021.100212

Cited by 14 publications

(11 citation statements)

References 47 publications

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“…SIM works by projecting a moving grid onto the sample and recording multiple images as this lattice moves, creating an interference pattern known as the Moiré effect [ 27 ]. Processing these images improves the resolution by a factor of two, and recently, further improvements to the image processing algorithm have enhanced this to an additional two-fold [ 28 ], providing a spatial resolution as low as 60 nm. An advantage of SIM technology is the use of digital cameras for detection, rather than single point detectors, enabling these microscopes to capture highly dynamic events at frame rates exceeding 100+ frames per second [ 29 ].…”

Section: Fluorescence Microscope Hardware Systemsmentioning

confidence: 99%

Fluorescence Microscopy—An Outline of Hardware, Biological Handling, and Fluorophore Considerations

Hickey

Ung

Bader

et al. 2021

Cells

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Fluorescence microscopy has become a critical tool for researchers to understand biological processes at the cellular level. Micrographs from fixed and live-cell imaging procedures feature in a plethora of scientific articles for the field of cell biology, but the complexities of fluorescence microscopy as an imaging tool can sometimes be overlooked or misunderstood. This review seeks to cover the three fundamental considerations when designing fluorescence microscopy experiments: (1) hardware availability; (2) amenability of biological models to fluorescence microscopy; and (3) suitability of imaging agents for intended applications. This review will help equip the reader to make judicious decisions when designing fluorescence microscopy experiments that deliver high-resolution and informative images for cell biology.

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Section: Fluorescence Microscope Hardware Systemsmentioning

confidence: 99%

Fluorescence Microscopy—An Outline of Hardware, Biological Handling, and Fluorophore Considerations

Hickey

Ung

Bader

et al. 2021

Cells

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“…Dehalogenases act as tags when genetically fused to a protein of interest, termed HaloTag technology (England et al, 2015;Döbber and Pohl, 2017;Erdmann et al, 2019). This technology overcomes the current limitations of traditional protein tagging platforms, as it can be applied to protein isolation and purification, studies of protein synthesis and degradation, analyses of protein function, studies of protein-protein and protein-DNA interactions, and molecular and cellular imaging (Encell et al, 2012;Merrill et al, 2019;Cattoglio et al, 2020;Freitas et al, 2021;Minner-Meinen et al, 2021). Furthermore, novel technologies have been developed for tumor diagnosis and treatment involving the linkage of dehalogenase fused with cancer cell recognition peptides to multifunctional nanoparticles (Garbujo et al, 2020).…”

Section: Other Fieldsmentioning

confidence: 99%

Mini Review: Advances in 2-Haloacid Dehalogenases

et al. 2021

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The 2-haloacid dehalogenases (EC 3.8.1.X) are industrially important enzymes that catalyze the cleavage of carbon–halogen bonds in 2-haloalkanoic acids, releasing halogen ions and producing corresponding 2-hydroxyl acids. These enzymes are of particular interest in environmental remediation and environmentally friendly synthesis of optically pure chiral compounds due to their ability to degrade a wide range of halogenated compounds with astonishing efficiency for enantiomer resolution. The 2-haloacid dehalogenases have been extensively studied with regard to their biochemical characterization, protein crystal structures, and catalytic mechanisms. This paper comprehensively reviews the source of isolation, classification, protein structures, reaction mechanisms, biochemical properties, and application of 2-haloacid dehalogenases; current trends and avenues for further development have also been included.

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“…Since its development and commercialization by Promega, HaloTag has become a valuable research tool for a broad range of applications (Figure A) including protein purification and immobilization, enhancement of the soluble expression of recombinant proteins, cellular protein imaging, , imaging in vivo, and single-molecule studies. − The technology is applicable to the analyses of protein–protein and protein–nucleic acid interactions, , proteome stress, , protein folding and aggregation, , dynamics and hydration, − or cell permeability . HaloTag fusions enable protein control in vivo, − including degradation , or dimerization of proteins of interest.…”

Section: Introductionmentioning

confidence: 99%

Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling

Marques

Slanska

Chmelova

et al. 2022

JACS Au

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HaloTag labeling technology has introduced unrivaled potential in protein chemistry and molecular and cellular biology. A wide variety of ligands have been developed to meet the specific needs of diverse applications, but only a single protein tag, DhaAHT, is routinely used for their incorporation. Following a systematic kinetic and computational analysis of different reporters, a tetramethylrhodamine- and three 4-stilbazolium-based fluorescent ligands, we showed that the mechanism of incorporating different ligands depends both on the binding step and the efficiency of the chemical reaction. By studying the different haloalkane dehalogenases DhaA, LinB, and DmmA, we found that the architecture of the access tunnels is critical for the kinetics of both steps and the ligand specificity. We showed that highly efficient labeling with specific ligands is achievable with natural dehalogenases. We propose a simple protocol for selecting the optimal protein tag for a specific ligand from the wide pool of available enzymes with diverse access tunnel architectures. The application of this protocol eliminates the need for expensive and laborious protein engineering.

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Split-HaloTag imaging assay for sophisticated microscopy of protein–protein interactions in planta

Cited by 14 publications

References 47 publications

Fluorescence Microscopy—An Outline of Hardware, Biological Handling, and Fluorophore Considerations

Fluorescence Microscopy—An Outline of Hardware, Biological Handling, and Fluorophore Considerations

Mini Review: Advances in 2-Haloacid Dehalogenases

Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling

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