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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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J. Bacteriol. 194, 1389-1400. Legionella pneumophila LidA affects nucleotide binding and activity of the host GTPase Rab1. 2012

Neunuebel, M.R., Mohammadi, S., Jarnik, M., and Machner, M.P.

Notes: In this study, the L. pneumophila protein Lem3 was expressed as a HaloTag® fusion protein and purified using HaloLink™ Resin. Lem3 was first cloned into the pFN22K HaloTag® Vector and the resultant HaloTag-Lem3 protein was expressed in Single-Step (KRX) competent cells before purification using the HaloTag® Protein Purification System. Lem3 was cleaved from the HaloLink™ Resin using TEV protease.


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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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Nat Chem. Biol. July 3, Epub. Small-molecule hydrophobic tagging-induced degradation of HaloTag fusion proteins. 2011

Neklesa, T.K., Tae, H.S., Schneekloth, A.R., Stulberg, M.J., Corson, T.W., Sundberg, T.B., Raina, K., Holley, S.A., and Crews, C.M.

Notes: These authors investigated whether HaloTag® technology could be used to specifically target and degrade intracellular proteins. They developed a method to degrade specific proteins of interest using a small molecule by tagging the HaloTag® protein with an adamantyl moiety. The hypothesis was that appending a hydrophobic residue to a protein surface domain would mimic partial denaturation and induce proteasomal degradation. They first used a HaloTag®-luciferase fusion protein to determine the biological activity of various small molecules that enhanced hydrophobicity. After selecting the small molecule that reduced luciferase activity in HEK293 cells, they went on to test their hypothesis in various in vitro and in vivo models. They were able to demonstrate degradation of various HaloTag®-linked transmembrane proteins expressed in HEK293 cells, to show degradations of target proteins in Zebrafish, and to show degradation of the HRAS oncogene product both in NIH3T3 cells and in a mouse tumor model. (4125)

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Protein Expr. Purif. 76, 154-164. HaloTag-based purification of functional human kinases from mammalian cells. 2010

Ohana, R.F., Hurst, R., Vidugiriene, J., Slater, M.R., Wood, K.V. and Urh, M.

Notes: The authors of this paper demonstrate the utility of the HaloTag® protein purification system for purifying functional proteins from mammalian cells. To this end five kinases were cloned into HaloTag® vectors, expressed in and purified from HEK293T cells. To demonstrate functionality of the purified recombinant kinases, activity was measured using the appropriate ADP-Glo™ Assay Kinase Enzyme Systems. (4145)

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Development 137, 901–11. SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification. 2010

Hattori, T., Müller, C., Gebhard, S., Bauer, E., Pausch, F., Schlund, B., Bösl, M.R., Hess, A., Surmann-Schmitt, C., von der Mark, H., de Crombrugghe, B. and von der Mark, K.

Notes: To study the role of the transcription factor Sox9 in the transition from cartilage to bone in newborn mice, the authors performed chromatin immunoprecipitation using the HaloCHIP™ System. Primary rib chondrocytes were transfected with a vector expressing full-length Sox9 with a HaloTag® protein tag, then proteins and DNA were cross-linked. DNA was isolated and sonicated, and the Sox9:DNA complexes were precipitated using the HaloLink™ Resin. The precipitated DNA then was amplified by PCR to determine that SOX9 binds to the SRY sites in the Vegfa gene. (4054)

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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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Protein Expr. Purif. 68(1), 110-120. HaloTag7: A genetically engineered tag that enhances bacterial expression of soluble proteins and improves protein purification 2009

Ohana, R.F., Encel, L.P., Zhao, K., Simpson, D., Slater, M.R., Urh, M., Wood, K.V.

Notes: The authors note that while many protein fusion tags are available to assist in purifying functional, recombinant proteins in soluble form and adequate amounts, none of the available tags are ideal when applied to the diversity of proteins studied. Available tags are often best-suited to specific aspects of the overall process, be it expression, solubilization, protein capture, etc. HaloTag was designed to address multiple features. The authors demonstrated that HaloTag provided enhanced expression and solubility in E. coli, with efficient protein purification and labeling for screening and quantitation. Enhanced solubility coupled with covalent capture gives HaloTag advantages over conventional affinity fusion tags, demonstrated by the successful purification of a wide variety of proteins, including low expressers, with higher yield and purity compared to the other tested tags. Covalent capture coupled with efficient tag removal to release the target protein, provides higher purity by eliminating tag contaminants. Using full-length cDNAs encoding 23 human proteins of varying size and function, the authors compared the efficacy of HaloTag as an expression and purification tag to the frequently used solubility enhancers MBP and GST. As reported previously, the three larger tags (HaloTag, MBP and GST) provided higher total and soluble expression compared to the smaller His6tag. In comparing soluble protein expression levels for the three larger tags, it was found that HaloTag solubilized 74% of the 23 human proteins examined, compared to 52% for MBP and 39% for GST. G2681 was one of the T7, bacterial promoter-based Flexi vectors used.

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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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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 microscopy. 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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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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Methods in Mol. Biol. 577, 25-39. High-Throughput Construction of ORF Clones for Production of the Recombinant Proteins 2009

Yamakawa, Hisashi

Notes: The authors use the Flexi® Cloning System to convert their cDNA clones to expression-ready clones. They wanted clones that could be used for comprehensive analysis with the HaloTag® Technology. They also describe a method of transferring ORFs between Flexi® Vectors in a 96-well plate format. They also used Wizard® SV 96 Plasmid DNA Purification, Wizard® SV PCR Clean-Up, and Wizard® SV Gel and PCR Clean-Up Systems. (4056)

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BMC Genomics 10, 497. A functional analysis of the CREB signaling pathway using 2009

Danette D Hartzell, Nathan D Trinklein, Jacqui Mendez, Nancy Murphy, Shelley F Aldred, Keith Wood and Marjeta Urh

Notes: The HaloCHIP System was used to measure CREB binding as an alternative to the ChIp method. (4057)

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J. Clin. Endocrinol. Metab. 94, 2594-2601. KLF15 Is a transcriptional regulator of the human 17beta-hydroxysteroid dehydrogenase type 5 gene. A potential link between regulation of testosterone production and fat stores in women 2009

Du, X., Rosenfield, R. and Qin, K.

Notes: The authors used a HaloTag® vector and the HaloCHIP™ System to identify a KLF15 binding site in the HSD17B5 promoter. (4059)

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J. Clin. Endocrinol. Metab. 94, 2650-2657. Posttranscriptional Regulation of CDC25A by BOLL Is a Conserved Fertility Mechanism Essential for Human Spermatogenesis 2009

Yung Ming Lin, Chia Ling Chung, and Yu Sheng Cheng

Notes: Human boule (BOLL) gene was subcloned into the HaloTag® pHT2 Vector and transformed into JM109 and HeLa cells. (4060)

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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-permeant and impermeant ligands and followed spatial separation of plasma membrane and internal pools of the integrin-HaloTag® protein. (3912)

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DNA Research 15, 137-149. Exploration of human ORFeome: High-throughput preparation of ORF clones and efficient characterization of their protein products. 2008

Nagase, T., Yamakawa, H., Tadokoro, S., Nakajima, D., Inoue, S., Yamaguchi, K., Itokawa, Y., Kikuno, R.F., Koga, H. and Ohara, O.

Notes: These authors used the Flexi® Vector System to prepare ORF clones encoding 1929 human genes and to transfer a subset of these clones to various expression vectors for further analysis. They created HaloTag® fusion proteins and examined expression of these proteins in vitro and in COS7 and HEK293 cells. They also performed comparisons between the Flexi® System and Gateway® cloning system, specifically examining the effects of flanking sequences on protein expression in in vitro translation systems and confirming that the cellular localization of the HaloTag® fusion proteins was consistent with results obtained using GFP-fusions. (3800)

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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 possesses 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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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 immobilization. The HaloTag® reporter protein is engineered to form covalent bonds with ligands that have different functional reporters. (3925)

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Appl. Environ. Microbiol. 73, 4234-4242. Rapid engineering of bacterial reporter gene fusions by using Red recombination. 2008

Gerlach, R.G., Hölzer, S.U., Jäckel, D., and Hensel, M.

Notes: These authors describe use of a red recombinase mediated method for generation of reporter constructs in Salmonella enterica setrovar typhimurium. Among the reporter constructs created was a HaloTag® reporter using the HaloTag® coding region from the pHT2 promoter. (3924)

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Mol. Biosyst. 4, 59-65. A general system for evaluating the efficiency of chromophore-assisted light inactivation (CALI) of proteins reveals Ru(II) tris-bipyridyl as an unusually efficient "warhead". 2008

Lee, J., Yu, P., Xiao, X. and Kodadek, T.

Notes: In this paper, researchers were looking for efficient chromophores for singlet oxygen generation used for chromophore-assisted light inactivation (CALI) of proteins. The HaloTag® protein and firefly luciferase were used to test how well the chromophores performed in crude extracts and living cells. The expression vector for an epitope-tagged Luciferase-HTP protein, 3X Flag-Luc-HTP-Myc, was constructed using firefly luciferase amplified from the pGL3-Basic Vector and HaloTag® (HTP) amplified from the pHT2 Vector. The fusion protein was tested for labeling with a HaloTag® biotin ligand by transfecting HeLa cells with 8μg of 3X Flag-Luc-HTP-Myc plasmid and 80ng of pRL-SV40 Vector. After transfection, cells were lysed with Passive Lysis Buffer and 2μl of HeLa cell lysate was diluted in 48μl of PBS + BSA and incubated for 30 minutes at room temperature with increasing concentrations of a biotin-HT ligand. The samples then were incubated with streptavidin-agarose for 30 minutes at room temperature, centrifuged and the luciferase activity of 20μl of supernatant was measured using the Dual-Luciferase® Reporter Assay System. The fusion protein was also tested using two chromophore ligands, ruthenium(II)tris-bipyridyl (Ru-HaloTag®[HT]) and fluoroscein-HT at a concentration of 100nM, and both were successful as measured by the Dual-Luciferase® Reporter Assay System. An in vivo CALI was performed by transfecting HeLa cells with 100ng of 3X Flag-HTP-Luc-Myc plasmid and 1ng of pRL-SV40 Vector for 15 hours, and treating the cells with Ru-HT or F-HT for 3 hours. The cells were then irradiated for 30 minutes, placed in the dark for 30 minutes, then the cells were lysed and analyzed with the DLR Assay. (3954)

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J. Proteome Res. 7(10), 4475-82. Improving protein array performance: focus on washing and storage conditions. 2008

Nath, N., Hurst, R., Hook, B., Meisenheimer, P., Zhao, K.Q., Nassif, N., Bulleit, R.F., and Storts, D.R.

Notes: These authors tested the effect of different washing, drying and storage conditions on the stability of various protein:protein interaction arrays. They tested five different interacting protein pairs and three enzymes, monitoring stability and activity under various processing conditions. They found that addition of 5% glycerol to the wash buffer helped retain enzyme activity during washing and drying. There was significant loss of enzyme activity when slides were stored dry at 4oC but slides stored at -20oC in 50% glycerol retained enzyme activity. HaloTag-fused enzymes in cell-free extracts could undergo multiple freeze-thaw cycles without significant effect on enzyme activity, indicating that such extracts could be frozen at -70oC and used to print small batches of arrays at various intervals over a period of weeks. (3890)

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