Hélène A Benink

Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate

Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ∼2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ∼150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.

In Vivo Labeling Method Using a Genetic Construct for Nanoscale Resolution Microscopy

We demonstrate beam scanning-stimulated emission depletion microscopy with in vivo labeled cells. A red emitting fluorescent dye is introduced into membrane protein fused to a multifunctional reporter protein (HaloTag, Promega, Madison, WI) in live cells. This approach allows superresolution stimulated emission depletion imaging without the limitations of immunofluorescence-based staining.

Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands.

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins.

Examining the complexity of human RNA polymerase complexes using HaloTag technology coupled to label free quantitative proteomics

Efficient determination of protein interactions and cellular localization remains a challenge in higher order eukaryotes and creates a need for robust technologies for functional proteomics studies. To address this, the HaloTag technology was developed for highly efficient and rapid isolation of intracellular complexes and correlative in vivo cellular imaging. Here we demonstrate the strength of this technology by simultaneous capture of human eukaryotic RNA polymerases (RNAP) I, II, and III using a shared subunit, POLR2H, fused to the HaloTag. Affinity purifications showed successful isolation, as determined using quantitative proteomics, of all RNAP core subunits, even at expression levels near endogenous. Transient known RNAP II interacting partners were identified as well as three previously uncharacterized interactors. These interactions were validated and further functionally characterized using cellular imaging. The multiple capabilities of the HaloTag technology demonstrate the ability to efficiently isolate highly challenging multiprotein complexes, discover new interactions, and characterize cellular localization.

Direct pH measurements by using subcellular targeting of 5(and 6-) carboxyseminaphthorhodafluor in mammalian cells

As a means of reliably measuring intracellular pH, we have precisely targeted 5(and 6-) carboxyseminaphthorhodafluor to cellular subcompartments. This was accomplished by combining the well-established pH-sensitive dye with a protein-based reporter system. When expressed in cells, the reporter protein is designed to covalently bind ligands composed of a functional group and a reactive linker. In order to make a pH-sensitive ligand, we chemically coupled the pH sensor to a reactive linker. Several ligands of differing linker lengths were made and tested for their pH responsiveness in vitro. The most responsive of these ligands was then evaluated for its efficacy in live cell labeling and its use as an intracellular pH sensor for ratiometric confocal microscopy. Here we show that we could target this pH sensor within mammalian cells exclusively to either the nucleus or cytoplasm. Exhibiting the versatility of this reporter technology, we were also able to specifically limit pH sensor labeling to within the trafficking pathway of integrins and directly measure pH of this environment. Results correspond well with previously published reports. Both the simplicity and flexibility of the technology used in this study make possible the development of diverse targeted microenvironmental sensors or other moieties of interest.

In Vivo Fluorescent Labeling of Tumor Cells with the HaloTag® Technology

Many fluorescent sensors are currently available for in vitro bio-physiological microscopic imaging. The ability to label cells in living animals with these fluorescent sensors would help translate some of these assays into in vivo applications. To achieve this goal, the first step is to establish a method for selectively labeling target cells with exogenous fluorophores. Here we tested whether the HaloTag® protein tagging system provides specific labeling of xenograft tumors in living animals. After systemic delivery of fluorophore-conjugated ligands, we performed whole animal planar fluorescent imaging to determine uptake in tag-expressing HCT116 xenografts. Our results demonstrate that HaloTag ligands containing red or near-infrared fluorophores have enhanced tumor uptake and are suitable for non-invasive in vivo imaging. Our proof-of-concept results establish feasibility for using HaloTag technology for bio-physiological imaging in living animals.

On-bead antibody-small molecule conjugation using high-capacity magnetic beads

Antibodies labeled with small molecules such as fluorophore, biotin or drugs play an important role in various areas of biological research, drug discovery and diagnostics. However, the majority of current methods for labeling antibodies is solution-based and has several limitations including the need for purified antibodies at high concentrations and multiple buffer exchange steps. In this study, a method (on-bead conjugation) is described that addresses these limitations by combining antibody purification and conjugation in a single workflow. This method uses high capacity-magnetic Protein A or Protein G beads to capture antibodies directly from cell media followed by conjugation with small molecules and elution of conjugated antibodies from the beads. High-capacity magnetic antibody capture beads are key to this method and were developed by combining porous and hydrophilic cellulose beads with oriented immobilization of Protein A and Protein G using HaloTag technology. With a variety of fluorophores it is shown that the on-bead conjugation method is compatible with both thiol- and amine-based chemistry. This method enables simple and rapid processing of multiple samples in parallel with high-efficiency antibody recovery. It is further shown that recovered antibodies are functional and compatible with downstream applications.

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast. In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1. These examples demonstrate the power of this technology in enabling the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics.

In vivo tracking of human neural progenitor cells in the rat brain using bioluminescence imaging.

BACKGROUND Stem cell therapies appear promising for treating certain neurodegenerative disorders and molecular imaging methods that track these cells in vivo could answer some key questions regarding their survival and migration. Bioluminescence imaging (BLI), which relies on luciferase expression in these cells, has been used for this purpose due to its high sensitivity. NEW METHOD In this study, we employ BLI to track luciferase-expressing human neural progenitor cells (hNPC(Luc2)) in the rat striatum long-term. RESULTS We show that hNPC(Luc2) are detectable in the rat striatum. Furthermore, we demonstrate that using this tracking method, surviving grafts can be detected in vivo for up to 12 weeks, while those that were rejected do not produce bioluminescence signal. We also demonstrate the ability to discern hNPC(Luc2) contralateral migration. COMPARISON WITH EXISTING METHODS Some of the advantages of BLI compared to other imaging methods used to track progenitor/stem cells include its sensitivity and specificity, low background signal and ability to distinguish surviving grafts from rejected ones over the long term while the blood-brain barrier remains intact. CONCLUSIONS These new findings may be useful in future preclinical applications developing cell-based treatments for neurodegenerative disorders.

Abstract 5522: X-MAN™ reporter cell lines: Enabling the study of endogenous promoter activity and protein dynamics.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC To allow promoter activity and protein dynamics to be studied at endogenous levels for the first time, Horizon Discovery has developed suites of X-MAN™ reporter cell lines incorporating NanoLuc™ and HaloTag® technologies from Promega. By creating these innovative reporters, we have removed the need for either exogenous plasmid based overexpression studies, or use of surrogate markers of activity, both of which can yield artefactual data. Horizons proprietary rAAV-based GENESIS™ gene editing platform has been used to introduce reporter genes (NanoLuc™ or HaloTag®) into several specific chromosomal loci (including HIF1A, cMYC, β-Catenin and NRF2) either as endogenous promoter fusions or in-frame protein fusions. Our extensive expertise in cell line engineering means that the technology can be rapidly applied to virtually any gene of interest. NanoLuc™ produces high intensity luminescence enabling accurate quantification of gene expression even at low endogenous expression levels. HaloTag® is a multifunctional protein reporter which can be used for many applications including intracellular fluorescent imaging of live cells in real time. Validation experiments, including kinetic measurements and treatment with compounds or conditions that modulate transcription or protein expression, reveal robust and reproducible results for all reporter cell lines and demonstrate their value in a wide variety of applications, from pathway analysis to high throughput screening platforms. To further demonstrate the utility of the X-MAN™ reporter cell lines in HTS-screening applications, we used the HCT116 HIF1A NanoLuc™ protein reporter line in a multiplexed siRNA library screen against 960 ‘druggable’ targets, under both normoxic and reduced oxygen conditions. As expected, many known regulators of HIF1A were identified, such as AKT, PDK1 and cRaf, showing once more the robust nature of the reporter system. Several novel regulators were also identified highlighting the value of the reporter cell lines for rapid identification of key regulators of endogenous proteins. In conclusion, we have used the combination of NanoLuc™, HaloTag® and the GENESIS™ gene editing platform to generate highly sensitive reporter cell lines that are capable of registering physiological levels of gene transcription and protein activity/localization in live cells. These reporter technologies can be used for a wide range of applications and provide an exciting new tool for biologically relevant drug discovery. Citation Format: Holly Astley, Suzanne Grooby, Jo Francis, Sue Griffin, Annette Little, Helene Benink, Jeff Kelly, Rebecca Foster. X-MAN™ reporter cell lines: Enabling the study of endogenous promoter activity and protein dynamics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5522. doi:10.1158/1538-7445.AM2013-5522