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Current Chemical Genomics 6, 72-78. HaloTag, a Platform Technology for Protein Analysis. 2012

Urh, M., and Rosenberg, M.

Notes: This paper provides an overview of the many applications of HaloTag® Technology. The authors describe the development of the technology, focusing on it's multifunctional utility for protein imaging, protein isolation and display, and in the study of protein complexes and interactions. They also discuss it's potential to facilitate proteomics research studies across complex biological systems at the biochemical, cell-based and whole animal level. (4325)

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Sci. Signal. 4(180), online. Identification of a lysosomal pathway that modulates glucocorticoid signaling and the inflammatory response 2011

Yuanzheng, H., Xu, Y., Zhang, C., Gao, X., Dykema, K.J., martin, K.R., Ke, J., Hudson, E.A., Khoo, S.K., Resau, J.H., Alberts, A.S., Mackeigan, J.P., Furge, K.A. and Xu, H.E.

Notes: Yuangheng He and colleagues asked how the weak alkaline compound chloroquine (CQ) enhances the anti-inflammatory effects of synthetic glucocorticoids like dexamethasone, which are used to treat a host of inflammatory and autoimmune diseases. In the process they explored the intersection of lysosomal degradation pathways and glucocorticoid receptor signaling. They used Dual-Glo® Luciferase Assay System to look at glucocorticoid receptor-mediated (GR) activation and repression of reporters in AD293 cells under a variety of conditions (presence or absence of CQ; stripped serum, loss of lysosome synthesis, inhibition of V-ATPase, etc). They also used HaloTag® protein fusions to observe the fate of GR populations in the presence or absence of CQ and in the presence or absence of compounds that impair proteasome function. Live-cell imaging of GR-HaloTag® protein fusions revealed a dynamic association of the GR with lysosomes. The authors showed that glucocorticoid signaling is regulated by lysosomes. (4203)

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Biochem. J. 436, 387–397. The novel Nrf2-interacting factor KAP1 regulates susceptibility to oxidative stress by promoting the Nrf2-mediated cytoprotective response. 2011

Maruyama, A., Nishikawa, K., Kawatani, Y., Mimura, J., Hosoya, T., Harada, N., Yamamato, M. and Itoh, K.

Notes: These authors first used a FLAG-tagged protein (nfr2) with a HeLa Nuclear extract and captured interacting proteins via SDS-PAGE and in-gel digests of bands to identify (Krüppel-associated box)-associated protein 1 (KAP1) as a potential interacting partner. Human KAP1 was purchased as a HaloTag® CMV Flexi® Vector from Kazusa and used in a Mammalian PullDown scenario (with HaloLink™ Resin) to demonstrate interaction between the two proteins. A reporter assay was used to show that KAP1 facilitates Nrf2 transactivation in a dose-dependent manner. The authors defined the interaction sites using GST-tagged nrf2 and various forms of KAP1-HaloTag® Fusions expressed in TNT® SP6 High-Yield Wheat Germ Extract. GST-tagged proteins were expressed in E. coli and bound to glutathione-Sepharose beads. These bound proteins were mixed with the KAP1 from the cell-free expression system, incubated for 4 hours at 4°C, washed and stained with the HaloTag® TMR Ligand for 30 minutes. The proteins from the pull-down assay were subjected to SDS-PAGE and the HaloTag® proteins detected by phosphorimaging and the GST proteins by Coomassie Brilliant Blue Staining. A two-hybrid system consisting of the pRL-TK Vector with a firefly luciferase reporter with Gal4 UAS, mouse Nrf-2 N-terminal domain and KAP1 was also used. The vectors were transfected into Nrf2 knockout MEFs for 4 hours then incubated for 36 hours before luciferase expression was determined using the Dual-Luciferase® Reporter Assay System. (4123)

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Chem. Commun. 27, 4040-42. Cell-based in vivo dual imaging probes using genetically expressed tags and chemical contrast agents. 2009

Hasegawa, Y., Honda, A., Umezawa, K., Niino, Y., Oka, K., Chiba, T., Aiso, S., Tanimoto, A., Citterio, D., and Suzuki, K.

Notes: These authors describe a method for cell-surface labeling with two genetically expressed tags, a biotin acceptor peptide (BAP) and HaloTag (HT). BAP and HT were fused with the N-terminus of the mouse Ig κ-chain leader sequence to direct them to the secretory pathway, and the c-terminus of the platelet-derived growth factor receptor transmembrane domain to anchor them to the plasma membrane. Fluorescent proteins were fused with BAP and HT to create the two distinct fluorescent transmembrance proteins BAP-YFP-TM and HT-CFP-TM. The constructs were introduced into HeLa cells and imaged simultaneously. The authors then demonstrated in vivo imaging by injecting the doubly-tagged HeLa cells into nude mice and imaging with near infrared fluorescent probes, and with magnetic resonance probes. (3998)

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Biophys. J. 96, L01-L03. In vivo labeling method using genetic construct for nanoscale resolution microscopy. 2009

Schröder, J. Benink, H., Dyba, M. and Los, G.V.

Notes: Traditionally light microscopy resolution has been limited by the diffraction of light. However several new technologies have emerged that partially overcome that limitation. One of these stimulated emission depletion (STED) microscopy is now commercially available and has been integrated into confocal microscope platforms. Because STED depends on fluorescent markers that fulfill specific spectroscopic needs, its uses have been limited. The authors of this paper demonstrate successful high resolution of β1-integrin-Halotag®-fusion protein distribution using STED microscopy. Use of the HaloTag® technology allows researchers to create a reporter that can be labeled with STED-optimized fluorescent tags. (3955)

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Anal. Biochem. 392, 45-53. Protein-protein interaction studies on protein arrays: effect of detection strategies on signal-to-background ratios. 2009

Hurst, R., Hook, B., Slater, M.R., Hatrnett, J., Storts, D.R., and Nath, N.

Notes: These authors compared 6 different detection strategies for protein-protein interactions on protein arrays. They expressed HaloTag® labeled bait proteins in a cell-free expression system, and captured these bait proteins onto coated glass slides using the HaloLink™ Array System. They then compared detection strategies using prey proteins labeled as follows: 1)35S methionine, 2) fluorescence (BODIPY-FL) and 3) biotin labeling of lysine residues using modified Lys tRNA, 4) chemical labeling after expression, 5) HaloTag® fusion, and 6) N-terminal FLAG tag. The authors evaluated signal:background ratios, adaptability to high-throughput screening, and ease of use. (3999)

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Methods in Mol. Biol. 577, 121-131. Pulse-Chase Experiment for the Analysis of Protein Stability in Cultured Mammalian Cells by Covalent Fluorescent Labeling of Fusion Proteins 2009

Kei Yamaguchi, Shinichi Inoue, Osama Ohara and Takahiro Nagase

Notes: The authors used the Halotag® Labeling Technology in pulse-chase experiments. They pulse labeled proteins in cultured mammalian cells. Using the HaloTag® Technology, they were able to monitor the degradation of the labeled protein, Smad1, that was induced by coexpression of Smurf1. They conclude that the HaloTag® Technology could be used to monitor the regulation of SMAD1 degradation. (4055)

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Proc. Natl. Acad. Sci. USA 106, 5669-5674. Regulation of the processivity and intracellular localization of Saccharomyces cerevisiae dynein by dynactin. 2009

Kardon, J.R., Reck-Peterson, S.L. and Vale, R.D.

Notes: These authors expressed recombinant dynactin and dynein in Saccharomyces cerevisiae and investigated their interactions in motility assays. They created a c-terminal Halotag-Dynactin fusion, and were able to site-specifically label the fusion protein with the fluorescent dye tetramethylrhodamine (TMR). They studied the effect of the purified dynactin fusion protein on the motility of dynein complexes using total internal reflection fluorescence micropscopy. Dynactin alone did not interact with microtubules. However, when coincubated with recombinant dynein, the TMR-labeled dynactin moved processively along microtubules. The authors then used truncation mutants of dynactin to identify the region of the dynactin molecule required for localization and enhanced processivity of dynein. (3960)

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Science 319, 294. Arabidopsis CLV3 peptide directly binds CLV1 ectodomain. 2008

Ogawa, M., Shinohara, H., Sakagami, Y. and Matsubayashi, Y.

Notes: The authors of this study investigated the role of the genes CLV1 and CLV3 stem cell renewal and differentiation in self-renewing shoot apical meristem (SAM) in Arabidopsis. CLV1 encodes a receptor kinase that is a negative regulator of cell growth, and therefore cannot be overexpressed in plant cells. CLV3, which encodes a protein that is processed to a 12-amino acid peptide, is believed to be a ligand for CLV1. In this study, the authors made a construct in which the CLV1 kinase domain was replaced with the HaloTag™ protein and expressed the fusion protein in tobacco BY-2 cells. The fusion protein was detected using the HaloTag™ TMR ligand. They next incubated membrane extracts from wildtype and transgenic BY-2 cells (expressing the HaloTag™-CLV1 fusion) with tritium-labeled CLV3. The transgenic cells showed significantly higher CLV3 binding than wildtype, and the authors show that CLV3 specifically labels a 130-kd band that corresponds to the HaloTag™-CLV1 fusion protein, indicating that CLV3 binds the ectodomain of CLV1. (3763)

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ACS Chemical Biology 3, 373–382. HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis 2008

Los, G.V., Encell, L.P., McDougall, M.G., Hartzell, D.D., Karassina, N., Zimprich, C., Wood, M.G., Learish, R., Ohana, R.F., Urh, M., Simpson, D., Mendez, J., Zimmerman, K., Otto, P., Vidugris, G., Zhu, J., Darzins, A., Klaubert, D.H., Bulleit, R.F., and Wood, K.V.

Notes: The authors of this study describe a reporter gene system that allows researchers to create one genetic construct that can be used for a variety of studies including imaging and protein imobilization. The HaloTag® reporter protein is engineered to form covalent bonds with ligands that have different functional reporters. (3925)

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J. Biol. Chem. 283, 11575-11585. Identification of ubiquitin ligase activity of RBCK1 and its inhibition by splice variant RBCK2 and protein kinase Cβ. 2008

Tatematsu, K., Yoshimoto, N., Okajima, T., Tanizawa, K., and Kuroda, S.

Notes: RBCK1 is a RING-IBR (ring in between ring fingers) protein previously shown to have transcriptional activity and to bind protein Kinase Cβ. This paper demonstrates that RBCK1 also posseses ubiquitin ligase E3 activity. Both FLAG-tag and HaloTag® labeled proteins were used to demonstrate this activity. To demonstrate the interaction between RBCK1 and ubiquitinated proteins, HaloTag®-ubiquitin and FLAG-RBCK1 were coexpressed in HEK293 cells in the presence or absence of a proteasome inhibitor. The anti-FLAG immunoprecipitates isolated from these cells were analyzed by SDS-PAGE and Western blotting using anti-HaloTag® and anti-FLAG antibodies and self-ubiquitination of RBCK1 was demonstrated. RBCK2, a splice variant of RBCK1 lacking the RING domain showed no self ubiquitination activity but was demonstrated to interact with RBCK1 in vivo and in vitro, and to inhibit the self-ubiquitination activity of RBCK1 in a FLAG-tag assay. Pulse-chase experiments, using HEK293 cells expressing HaloTag®-RBCK1 with or without RBCK2 and treated with HaloTag®-TMR ligand, were used to show that the half-life of RBCK1 was extended by overexpression of RBCK2. (3874)

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BMC Cell Biology 9:17, doi:10.1186/1471-2121/9/17. Spatial separation and bidirectional trafficking of proteins using a multi-functional reporter. 2008

Svendsen, S., Zimprich, C., McDougall, M.G., Klaubert, D.H., and Los, G.V.

Notes: This paper demonstrates use of HaloTag® technology to study expression, trafficking and translocation of an integrin-HaloTag® fusion protein. The authors fused the Halotag reporter protein to truncated integrin. They then labeled live cells with different cell-permaent and impermeant ligands and followed spatial separation of plasma membrane and internal pools of the integrin-HaloTag® protein. (3912)

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Plant J. 52, 175–184. Functional immobilization of plant receptor-like kinase onto microbeads towards receptor array construction and receptor-based ligand fishing. 2007

Shinohara, H. and Matsubayashi, Y.

Notes: The authors were looking for a method for ligand fishing experiments with plant receptor-like kinases (RLKs). The strategy chosen, functional immobilization on microbeads, used phytosulfokine (PSK) and its carrot cell receptor (DcPSKR1) as the test ligand-receptor pair. DcPSKR1 was cloned into the pHT2 HaloTag® Vector, adding the HaloTag® gene to the C terminus of DcPSKR1 or replacing the DcPSKR1 kinase domain. These constructs were transfected into BY-2 cells and expression confirmed by immunoblotting the membrane fraction and staining with DcPSKR1 and Anti-HaloTag antibodies. To confirm activity of the individual proteins, membrane fractions of BY-2 cells expressing the DcPSKR1-HaloTag® fusion proteins were tested for PSK binding activity to then HaloTag® binding activity confirmed using the HaloTag® TMR Ligand. The DcPSKR1-ΔKD-Halo protein was immobilized on HaloLink™ Resin and the Kd measured. The binding of PSK to the immobilized DcPSKR1-ΔKD-Halo protein was visualized by using fluorescently labeled Alexa488-PSK. Columns of HaloLink™ Resin with bound DcPSKR1-ΔKD-Halo protein were used to bind PSK ligands at physiological concentration, elute the ligands with a high-salt buffer and analyzed on LC-MS. (3946)

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J. Biol. Chem. 282, 13059-13072. XAF1 mediates tumor necrosis factor-α-induced apoptosis and X-linked inhibitor of apoptosis cleavage by acting through the mitochondrial pathway 2007

Straszewshi-Chavez, A., Visintin, I.P., Karassina, N., Los, G., Liston, P., Halaban, R., Fadiel, A. and Mor, G.

Notes: The authors sought to determine the mechanism by which first-trimester trophoblasts resist FAS ligand-induced apoptosis but remain sensitive to TNFα-mediated apoptosis. First trimester trophoblasts express XAF1 [X-linked inhibitor of apoptosis (XIAP)-associated factor 1], which may be involved in regulating their response to proapoptotic signals. The authors created HaloTag™-XAF1 fusion constructs and transiently transfected the first trimester trophoblast cell line (3A). Cells were labeled with the HaloTag™ TMR ligand, and XAF1 was shown to localize to the cytoplasm. 3A cells transiently transfected with the fusion construct were also separated into cytoplasmic and mitochondrial fractions. The fractions were labeled with HaloTag™ TMR ligand. Expression of the fusion peaked at 48 hours after transfection in both mitochondrial and cytoplasmic fractions. TNFα-treatment of 3A cells induced translocation of endogenous XAF1 to the mitochondria. The authors used the Caspase-Glo® Assays to demonstrate activation of caspase-3 and caspase-9 in response to expression of XAF-1. They also show that caspase-3 activation and XIAP cleavage correlate with translocation of endogenous XAF1 to mitochondria. Viability of 3A and primary trophoblasts over expressing XAF1 was evaluated using the CellTiter® 96 AQueous One-Solution Assay. (3760)

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J. Exp. Bot. July, epub ahead of print. HaloTag™: A new versatile reporter gene system in plant cells. 2006

Lang, C., Schulze, J., Mendel, R-R. and Hänsch, R.

Notes: This paper highlights the first use of the HaloTag™ Interchangeable Protein Labeling Technology in plant cells. The cDNA for the HaloTag™ protein was amplified by PCR from the pHT2 Vector and cloned into the pGEM®-T Easy® Vector, from which it was excised and transferred to a second vector where its expression was under the control of the cauliflower mosaic virus (CaMV)-35S promoter. The construct was transformed into tobacco protoplasts and bioballistically transformed into tobacco leaf cells. Localization was followed using the HaloTag™ TMR and diAcFAM Ligands. (3503)

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Cell 126, 335-348. Single-molecule analysis of dynein processivity and stepping behavior. 2006

Reck-Peterson, S.L., Yidiz, A., Carter, A.P., Gennerich, A., Zhang, N. and Vale, R.D.

Notes: HaloTag™ Interchangeable Labeling Technology was used to specifically label engineered dynein produced in Saccharomyces cerevisiae. The HaloTag™ Protein was added in-frame with either the 5´ or 3´ end of the coding sequence of various engineered dynein molecules. The authors report being able to label the dynein in specific locations using fluorescent dyes or quantum dots. HaloTag™ TMR Ligand was used to covalently label dynein to directly visualize dynein motor movement using total internal reflection fluorescence microscopy. HaloTag™ Biotin Ligand was used to label dynein with streptavidin quantum dots. (3504)

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