Selective protein visualization through creation of genetic fusions is a powerful analytical method commonly used to elucidate protein function in complex biological environments such as cells or organisms. Unlike GFP, HaloTag and HaloTag-POI fusions are per se not fluorescent, but rather they only become fluorescent upon the addition of a fluorescent HaloTag ligand. This is an important distinction as it gives spatial and temporal control of labeling. The process of fluorescent labeling is simple and rapid requiring only the addition of the fluorescent HaloTag ligand to the media for a brief 15-30 min incubation followed by a few quick washes. Wash steps can be avoided by using an optimized concentration of the ligand such as HaloTag R110Direct ligand. This ligand is simply added to the cell media at the time of plating of the cells. The labeling on demand aspect of HaloTag fusions allows for development of unique applications such as pulse chase and differential spatial labeling of proteins. Traditionally, metabolic pulse-chase with isotopically labeled amino acids has been used to monitor stability and synthesis of specific proteins in cells [30Dickson RC, Mendenhall MD, Eds. Signal transduction protocols Totova NJ: Humana Press 2004.]. The low temporal resolution of this method limits the use of this approach especially when studying proteins with relatively long half-lives. The ability to rapidly label proteins at a particular time point in combination with availability of different fluorescent ligands makes HaloTag particularly suitable for pulse chase based analysis of protein turnover [31He Y, Xu Y Zhang, et al. Identification of a lysosomal pathway that modulates glucocorticoid signaling and the inflammatory Sci Signal 2011; 4: 180-8.]. In a typical experiment a HaloTag-protein fusion is labeled with one of the available fluorescent ligands (e.g. green), followed by removal of this ligand by media exchange and addition of a ligand with a different color (e.g. red). The second ligand is present in the media throughout the duration of the experiment and will specifically label all newly synthesized protein in red, while the old protein remains green. As a result of this process the two pools of protein can be distinguished allowing analysis of degradation and synthesis. Importantly, it is the covalent binding property of HaloTag ligands that imparts long-term fluorescent labeling and prevents dissociation of the first bound ligand from the labeled protein while subsequent labeling is occurring with the second added ligand. The rapid binding kinetics is also important since it allows labeling of cellular proteins in minutes and ensures complete labeling of low abundant proteins, which is critical during the short pulse labeling phase. Without these performance attributes of HaloTag, the complete labeling of the first protein pool would be compromised.

Dual labeling using different fluorescent ligands can also be used if studying membrane bound versus intracellular protein populations. In this approach the different color ligands also differ in regards to permeability. By combining a cell permeable ligand in one color with a non-permeable ligand of different color we can differentially label surface exposed proteins where HaloTag is exposed on the cellular surface, from the intracellular pool of the same protein. This affords analysis of protein trafficking, endocytosis and study of membrane protein biology [32Svendsen S, Zimprich C, McDougall MG, Klaubert DH, Los GV. Spatial separation and bidirectional trafficking of proteins using a multi-functional reporter BMC Cell Biol 2008; 9: 7-31.].

Other imaging applications such as super-resolution imaging and single molecule imaging approaches have also adopted HaloTag technology [24Lee HL, Lord SJ, Iwanaga S, et al. Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores J Am Chem Soc 2010; 132 (43 ): 15099-01., 33Wilmes S, Staufenbiel M, Lisse D, et al. Triple-color super-resolution imaging of live cells: resolving submicroscopic receptor organization in the plasma membrane Angew Chem Int Ed Eng 2012; 51(20 ): 4868-71.-36Schroder J, Benink H, Dyba M, Los GV. In vivo labeling method using a genetic construct for nanoscale Biophys J 2009; 96 (Issue 1 ): L1-3.]. In one of these studies the observation was made that the relatively hydrophobic nature of HaloTag ligand appeared to support more efficient permeation of the super-resolution dye (ATTO655) when coupled to the HaloTag reactive ligand using the N-hydroxyl succinimidyl chemistry [33Wilmes S, Staufenbiel M, Lisse D, et al. Triple-color super-resolution imaging of live cells: resolving submicroscopic receptor organization in the plasma membrane Angew Chem Int Ed Eng 2012; 51(20 ): 4868-71.]. Combination of pho-toactivatable fluorescent proteins and HaloTag-ATTO655 allowed for multi-color super-resolution imaging of live cells and submicroscopic resolution of receptor organization in the plasma membrane. HaloTag imaging can also provide the means of reliably measuring intracellular pH by precise targeting of 5(and 6-) carboxyseminaphthorhodafluor (SNARF-1) to sub-cellular compartments. This was accomplished by coupling SNARF-1 to the HaloTag reactive ligand and by generating HaloTag fusions with proteins which reside in specific sub-cellular locations such as nucleus or cytoplasm [37Benink HA, McDougall MG, Klaubert DH, Los GV. Direct pH measurements by using subcellular targeting of 5(and 6-) carboxyseminaphthorhodafluor in mammalian cells BioTech 2009; 47: 69-74.]. The localization of HaloTag protein fusions is dictated by the fusion partner allowing targeted positioning of HaloTag protein to different cellular compartments. Subsequently, cells are treated with the HaloTag ligand carrying the pH sensor dye (SNARF) which binds to HaloTag protein localized to a particular location in a cell. Depending on the pH of the environment the fluorescent signal of SNARF changes allowing discernible measurement of pH in different locations.

The simplicity and flexibility of the technology makes this system amenable for development of other imaging applications and recently, in vivo animal imaging using HaloTag has been reported [26Strauch RC, Mastarone DJ, Sukerkar PA, Song Y, Ipsaro JJ, Meade TJ. Reporter protein-targeted probes for magnetic resonance imaging J Am Chem Soc 2011; 133 (41 ): 16346-9., 28Hong H, Benink HA, Zhang Y, Yang Y, Uyeda TH, Engle JW. HaloTag: a novel reporter gene for positron emission tomography Am J Transl Res 2011; 3(4 ): 392-403., 38Kosaka N, Ogawa M, Choyke PL, Karassina N, Corona C, McDougall M. In vivo stable tumor-specific painting in various colors using Dehalogenase-Based Protein-Tag fluorescent ligands Boiconjug Chem 2009; 20: 1367-74.]. In these studies in vivo imaging in animal models expressing HaloTag fusions on the surface of tumorigenic cells were performed allowing detection of growing tumors upon injection into animals using a variety of different fluorescent or PET HaloTag ligands.

As noted earlier HaloTag is a powerful tool for purification of proteins, isolation of cellular protein complexes (protein-protein and protein-DNA), and for protein capture and display. To utilize HaloTag for direct protein purification, a chromatographic matrix carrying the chloroalkane ligand (HaloLink resin) was created. The covalent nature of the HaloTag-fusion protein capture combined with rapid binding kinetics overcomes the equilibrium-based limitations associated with traditional non-covalent affinity purification and enables efficient capture of target proteins even at very low abundance. Furthermore, it allows for extensive and stringent washing without loss of bound protein due to diffusion. While the covalent association clearly has its advantages it also creates a challenge in eluting the protein of interest. As the covalent bond between HaloTag and chloroalkane cannot be readily reversed, traditional approaches to elute proteins from the resin cannot be utilized. Instead the protein of interest can be released by specific protease (TEV) cleavage, as the TEV protease recognition site is present between the HaloTag and the fusion partner. Upon cleavage, HaloTag stays bound to the resin while the fusion partner is released, yielding highly pure protein free of tag. It is important to note that a few amino acids of the linker segment connecting the two proteins remain associated with the protein after the cleavage. Depending on the N or C orientation of the HaloTag, 6 or 13 amino acids of the linker sequence, respectively, remain with the purified protein.

Successful purification of functional proteins generally requires efficient expression of recombinant proteins in soluble form. This is especially a challenge when heterologous or large proteins are expressed in E. coli, one of the most frequently used expression hosts due to easy manipulation, rapid growth and low cost [39Baneyx F. Recombinant protein expression in Escherichia coli Curr Opin Biotechnol 1999; 10: 411-22.-41Braun P, Hu Y, Shen B, et al. Proteome-scale purification of human proteins from bacteria Proc Natl Acad Sci USA 2002; 99: 2654-9.]. One approach to overcome the limitations of low expression of soluble protein is to optimize expression conditions such as temperature, growth media, induction conditions etc. To simplify the screening of these variables, fluorescent tags such as GFP have been utilized [42Waldo GS. Improving protein folding efficiency by directed evolution using the GFP folding reporter Methods Mol Biol 2003; 230: 343-59., 43Omoya K, Kato Z, Matsukuma E, et al. Systematic optimization of active protein expression using GFP as a folding reporter Protein Expr Purif 2004; 36: 327-.]. HaloTag offers an excellent alternative not only because it is an excellent purification tool, but also because it allows for easy fluorescent detection and quantitation of expressed proteins. Furthermore, unlike GFP, the HaloTag fluorescent signal survives boiling and SDS-PAGE gel electrophoresis and therefore allows for gel based resolution and quantification using the fluorescent signal. Thus, HaloTag can be used for rapid screening and purification. Since HaloTag protein labeled with fluorescent HaloTag ligand can no longer bind to the resin, the labeling reaction is performed on small aliquots of the sample. The utility and superior performance of this system for purification of proteins from E. coli has been demonstrated [44Friedman OR, Encell LP, Zhao K, et al. Halo Tag7: a genetically engineered Tag that enhances bacterial expression of soluble proteins and improves protein purification Protein Expr Purif 2009; 68(1 ): 110-20.]. In the same report the authors also note that HaloTag improves the solubility of the fusion proteins.

There is an increasing need and desire to express and purify mammalian derived proteins directly from cultured mammalian cells retaining the physiological relevance and providing a more compatible environment for producing properly folded and functional mammalian proteins. Mammalian cells possess the proper machinery to produce mature proteins through proteolytic processing and are able to carry out relevant post–translational modification (PTM) which many proteins require for biological activity [45Cazalla D, Sanford JR, Caceres JF. A rapid and efficient protocol to purify biologically active recombinant proteins from mammalian cells Protein Expr Purif 2005; 42: 54-8.-47Wurm FM. Production of recombinant protein therapeutics in cultivated mammalian cells Nat Biotechnol 2004; 22: 1393-8.]. Therefore, achieving satisfactory purification of proteins from mammalian cells is of high interest and importance. Unfortunately, many of the traditional affinity tags that have been developed, fail to adequately purify proteins from mammalian cells. This is often due to inefficient binding of the tagged proteins to their purification matrices at lower protein expression levels typically observed in mammalian cells, as well as the nonspecific binding of endogenous proteins with similar affinities resulting in lower yields and purity of recovered proteins [48Pham PL, Perret S, Doan HC, et al. Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency Biotechnol Bioeng 2003; 84: 332-42.-50Lichty JJ, Malecki JL, Agnew HD, Michelson-Horowitz DJ, Tan S. Comparison of affinity tags for protein purification Protein Exp Purif 2005; 41: 98-105.]. These limitations are reduced significantly when HaloTag is used for purification of proteins from mammalian cells. This is due to the performance attributes of HaloTag described above; the high binding rate, the covalent binding and TEV specific release all work in concert to improve protein recovery, purity and yield. Absence of proteins analogous to HaloTag in mammalian cells also contributes to improved purity of proteins purified via HaloTag. Moreover, HaloTag fluorescent detection can be used for easy evaluation of optimization of expression and parameters such as transfection conditions, culturing conditions, expression clone selection, etc. [51Ohana RF, Hurst R, Vidugiriene J, Slater MR, Wood KV, Urh M. HaloTag-based purification of functional human kinases from mammalian cells Protein Expr Purif 2011; 76(2 ): 54-64., 52Chumanov RS, Kuhn PA, Xu Wei, Burgess RR. Expression and purification of full-lengthmouseCARM1 from transiently transfected HEK293T cells using HaloTag technology Protein Exp Purif 2011; 760: 145-53.].

Protein interactions, protein complexes and networks remain the focal point of current studies in cellular biology. Significant effort has been focused on analysis of global protein network analysis and genome mapping. Trapping both transient and often weak protein interactions presents a significant challenge as dissociation occurs before complexes can be adequately captured. The same properties which benefit HaloTag-based purification also make HaloTag especially well suited for capture of protein complexes directly from mammalian cells. Rapid and highly specific capture of HaloTag eliminates the need for long binding incubations and extensive washing thereby increasing the chance of recovery of transient interactions. With this approach a number of known transiently interacting partners of RNAP II were identified, in addition to three previously uncharacterized interacting proteins [53Daniels DL, Méndez J, Mosley AL, et al. Examining the complexity of human RNA polymerase complexes using HaloTag technology coupled to label free quantitative proteomics J Proteome Res 2012; 11 (2 ): 564-75.]. A similar approach was also used to capture cross-linked protein-DNA complexes providing an antibody free alternative to ChIP analysis [54Hartzell DD, Trinklein ND, Mendez J, et al. A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays BMC Genom 2009; 10: 497-512.].

Importantly, the same HaloTag fusion used for isolation of various protein complexes can also be used for covalent capture and surface display and further confirmation, validation or discovery of new protein interactions. The system was uniquely designed for allowing specific capture of the proteins onto glass slides (HaloLink protein arrays system) and other surfaces directly from lysates, thus alleviating the need for tedious protein purification, a major bottleneck in fabrication of current protein arrays.

The advantage of this system is that unlike other covalent immobilization techniques, where binding of protein is through multiple attachment sites and leads to random, often improper orientation, immobilization using HaloTag is a single point attachment through its active site ligand and therefore provides uniform orientation of the POI. Single point, oriented attachment increases capacity, effectiveness and reproducibility of the system preventing surface induced protein unfolding and improving protein activity and native conformation [55Nath N, Hurst R, Hook B, et al. Improving protein array performance: focus on washing and storage conditions J Proteome Res 2008; 10: 4475-82.]. The need for protein specific optimization of capture conditions is also eliminated as the immobilization relies on the same chemistry, namely attachment of HaloTag to the HaloTag ligand-modified surface. This simplifies the process, and significantly improves the uniformity of the arrays and reproducibility of the assays. The rapid preparation of HaloTag-fusion arrays and excellent performance opens-up the possibility for screening hundreds or thousands of different protein interactions [56Hurst R, Hook B, Slater MR, Hartnett J, Storts DR, Nath N. Protein-protein interaction studies on protein arrays: effect of detection strategies on signal-to-background ratios Anal Biochem 2009; 392(1 ): 45-53., 57Peterson SN, Kwon K. Application of a novel HaloTag to characterize protein-protein and protein-DNA interactions Curr Chem Genom 2012; 6: 8-17.].

The various applications and analyses described above are invaluable for studies directed at the elucidation of protein function. Such studies are often complemented by approaches that attempt to achieve specific disruption/ elimination of the activity of proteins in an effort to understand the underlying mechanisms governing a particular biological system. Historically, methods such as genetic knock-out technology, mutagenesis, antisense RNA and RNA interference (RNAi) have been used to specifically alter or eliminate a function of a particular protein. However, these methods are all indirect effectors of the POI and suffer from genetic compensation, low time resolution and can be difficult to use in live animals. HaloTag provides a new analytical tool for specific and direct protein depletion or inactivation of protein function at the protein level [27Takemoto K, Matsuda T, McDougall M, Klaubert DH, Hasegawa AGV. Chromophore-assisted light inactivation of HaloTag fusion proteins labeled with eosin in living cells ACS Chem Biol 2011; 6(5 ): 401-6., 29Neklesa TK, Tae HS, Schneekloth AR, Stulberg MJ, Corson TW, Sundberg TB. Small-molecule hydrophobic tagging-induced degradation of HaloTag fusion proteins Nat Chem Biol 2011; 7: 538-43., 58Tae HS, Sundberg TB, Neklesa TK, et al. Identification of hydrophobic tags for the degradation of stabilized proteins Chem Bio Chem 2012; 13: 538-41., 59Lee HD, Lord SJ, Iwanaga S, et al. Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores J Am Chem Soc 2010; 132 (43 ): 15099-01.]. Studies showed that binding of HaloTag ligands carrying a hydrophobic moiety to HaloTag fusion proteins destines these proteins for effective proteosomal degradation. This novel hydrophobic tagging technology of HaloTag fusions efficiently induced degradation of proteins in cell culture systems, as well as directly in animals, potentially making this system ideal for validating potential drug targets in disease models [29Neklesa TK, Tae HS, Schneekloth AR, Stulberg MJ, Corson TW, Sundberg TB. Small-molecule hydrophobic tagging-induced degradation of HaloTag fusion proteins Nat Chem Biol 2011; 7: 538-43., 58Tae HS, Sundberg TB, Neklesa TK, et al. Identification of hydrophobic tags for the degradation of stabilized proteins Chem Bio Chem 2012; 13: 538-41.].

Clearly, one major effort of biological research is to understand protein function and the interactions which regulate the majority of cellular processes. Development of new tools enabling a more comprehensive analysis of proteins is essential for furthering our understanding of this complex field. Here we presented a powerful platform, based on HaloTag technology, which offers both the reality and future promise of overcoming many of the limitations of current protein analysis approaches.