HaloTag Technology: A Versatile Platform for Biomedical Applications

England, Christopher G.; Luo, Haiming; Cai, Weibo

doi:10.1021/acs.bioconjchem.5b00191

Cited by 254 publications

(224 citation statements)

References 131 publications

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“…Because green fluorescent protein (GFP) imported into the mitosome matrix is not fluorescent [23], GFP-tagging of mitosomes has not been possible until now. Martinkova et al [59] have shown that mitosomes in trophozoites can be labeled for live cell microscopy using HaloTag markers [60]. However, no quantitative information on the spatial dynamics of peripheral mitosomes in the cytoplasm was presented in this report.…”

Section: Resultsmentioning

confidence: 92%

An Interactome-Centered Protein Discovery Approach Reveals Novel Components Involved in Mitosome Function and Homeostasis in Giardia lamblia

et al. 2016

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Protozoan parasites of the genus Giardia are highly prevalent globally, and infect a wide range of vertebrate hosts including humans, with proliferation and pathology restricted to the small intestine. This narrow ecological specialization entailed extensive structural and functional adaptations during host-parasite co-evolution. An example is the streamlined mitosomal proteome with iron-sulphur protein maturation as the only biochemical pathway clearly associated with this organelle. Here, we applied techniques in microscopy and protein biochemistry to investigate the mitosomal membrane proteome in association to mitosome homeostasis. Live cell imaging revealed a highly immobilized array of 30–40 physically distinct mitosome organelles in trophozoites. We provide direct evidence for the single giardial dynamin-related protein as a contributor to mitosomal morphogenesis and homeostasis. To overcome inherent limitations that have hitherto severely hampered the characterization of these unique organelles we applied a novel interaction-based proteome discovery strategy using forward and reverse protein co-immunoprecipitation. This allowed generation of organelle proteome data strictly in a protein-protein interaction context. We built an initial Tom40-centered outer membrane interactome by co-immunoprecipitation experiments, identifying small GTPases, factors with dual mitosome and endoplasmic reticulum (ER) distribution, as well as novel matrix proteins. Through iterative expansion of this protein-protein interaction network, we were able to i) significantly extend this interaction-based mitosomal proteome to include other membrane-associated proteins with possible roles in mitosome morphogenesis and connection to other subcellular compartments, and ii) identify novel matrix proteins which may shed light on mitosome-associated metabolic functions other than Fe-S cluster biogenesis. Functional analysis also revealed conceptual conservation of protein translocation despite the massive divergence and reduction of protein import machinery in Giardia mitosomes.

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Section: Resultsmentioning

confidence: 92%

An Interactome-Centered Protein Discovery Approach Reveals Novel Components Involved in Mitosome Function and Homeostasis in Giardia lamblia

et al. 2016

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“…Another novel technology called the HaloTag fusion tag, allowed for rapid evaluation of several potential fluorophores. 47 The HaloTag system is based on the formation of a stable fusion protein that carries an active site for binding of application-specific ligands. This results in a covalent interaction between the HaloTag-tagged protein and specific ligand.…”

Section: Applications Of the Nanoluc Technologymentioning

confidence: 99%

NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence

2016

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The biomedical field has greatly benefited from the discovery of bioluminescent proteins. Currently, scientists employ bioluminescent systems for numerous biomedical applications, ranging from highly sensitive cellular assays to bioluminescence-based molecular imaging. Traditionally, these systems are based on Firefly and Renilla luciferases; however, the applicability of these enzymes is limited by their size, stability, and luminescence efficiency. NanoLuc (NLuc), a novel bioluminescence platform, offers several advantages over established systems, including enhanced stability, smaller size, and >150-fold increase in luminescence. In addition, the substrate for NLuc displays enhanced stability and lower background activity, opening up new possibilities in the field of bioluminescence imaging. The NLuc system is incredibly versatile and may be utilized for a wide array of applications. The increased sensitivity, high stability, and small size of the NLuc system have the potential to drastically change the field of reporter assays in the future. However, as with all such technology, NLuc has limitations (including a non-ideal emission for in vivo applications and its unique substrate) which may cause it to find restricted use in certain areas of molecular biology. As this unique technology continues to broaden, NLuc may have a significant impact in both preclinical and clinical fields, with potential roles in disease detection, molecular imaging, and therapeutic monitoring. This review will present the NLuc technology to the scientific community in a non-biased manner, allowing the audience to adopt their own views of this novel system.

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“…So far, the HaloTag is mainly used for various biomedical applications, e.g. the in vivo detection of protein interactions and cell imaging (Los et al, 2008;England et al, 2015). In 2010, an initial study of this tag for biocatalysis was published using a monomeric lipase from Bacillus thermocatenulatus bound to magnetic beads.…”

Section: Introductionmentioning

confidence: 99%