Several criteria were considered during the development of HaloTag to ensure optimal performance and broad utility of HaloTag-POI fusions. Important considerations included: the reversible or irreversible nature of attachment of chemical functionalities (ligands) to the protein, high specificity of ligand binding, ability to perform reactions in vitro, in living cells and ideally also in whole animals. In addition, we focused on particular attributes of HaloTag ligands that differentiate them from other systems, such as the highly efficient reactivity with protein even at low protein concentration within timescales compatible with biological processes. Many different chemical functionalities (HaloTag ligands) have now been created by us and others, without affecting the specificity and kinetics of binding, ensuring that the ligands are not toxic to the cell and, when appropriate, cell permeable. In order to enable broad utility and easy adoption, most of these ligands are commercially available, easy-to-use, and do not require any special knowledge of chemistry synthesis.
The considerations described above played a critical role in our selection of the system that was used for development of HaloTag. We chose the bacterial haloalkane dehalgenase, because it exhibits features that allowed us to develop a tag with all the desired attributes. A brief summary of the development and optimization is described below; a more complete description of the evolution of HaloTag can be found in work by Los and Encell [20
The bacterial dehalogenase catalyzes a reaction that forms a covalent transition state intermediate with the chloroalkane substrate. This covalent bond is subsequently hydrolyzed, releasing the substrate and regenerating the active site [20
]. It was previously shown that mutating a histidine residue in the active site of the dehalogenase from Xanthobacter
prevented hydrolysis of the covalent bond, leading to formation of a irreversible bond with the substrate [23
]. By applying an analogous mutation to the Rhodococcus
dehalogenase we showed that an irreversible (covalent) bond can readily be formed with chloroalkane derivatives. The protein carrying this mutation was then called HaloTag and the chloroalkane derivatives, HaloTag ligands [20
]. Together they allowed for development of a system where HaloTag ligands carrying different functional groups could be permanently associated with the protein thereby imparting many different functionalities onto the protein.
The fact that dehalogenase is monomeric, relatively small (33 kDa) and that neither eukaryotic cells nor most bacteria (e.g. E. coli
) possess the haloalkane dehalogenase or its ligands provide excellent advantages. There is no endogenous equivalent in any of the standardly used experimental biological systems which would compete for binding of Halo Tag ligands and interfere with its specificity and efficiency. However, certain important performance attributes were still lacking in the original HaloTag variant. For example, the slow kinetics of substrate binding of HaloTag posed a significant limitation. In an effort to improve the properties of HaloTag, it was subjected to several rounds of site specific and random mutagenesis. The result was a HaloTag exhibiting a rapid substrate binding rate, comparable to that of tetrameric streptavidin with biotin [20
]. This protein engineering process also improved the thermostability and solubility properties of HaloTag. The resulting HaloTag protein displayed rapid, specific and covalent binding to the HaloTag ligands and allowed for expression of highly soluble fusion proteins in the absence of any endogenous competitors in essentially any biological system of interest.
As noted above, all HaloTag ligands comprise the same chloroalkane based binding group, onto which a variety of functional groups are appended using standard chemical synthesis. A series of fluorescent HaloTag ligands were developed by coupling TMR, Oregon Green, Coumarin, Alexa Fluor® 488, Alexa Fluor® 660 and R110 dyes with the reactive binding group. The resulting ligands readily permeate into mammalian cells, with exception of the Alexa Fluor 488 substrate, which is an important cell impermeable variant. Importantly, at the appropriate concentrations these ligands show no cytotoxicity. Thus, with these fluorescent dyes it is now possible to make any HaloTag-POI fusion permanently fluorescent and, in addition, by selecting different dyes it is possible to label the HaloTag fusion with different fluorescent dye in different experiments or even at different time points within the same experiment.
To enable applications beyond fluorescent labeling, such as protein capture and display, HaloTag ligand surfaces were generated. Significant efforts were dedicated towards development of surfaces with very low nonspecific binding and efficient specific binding of HaloTag fusion proteins. Currently three formats of surfaces, magnetic resin, non-magnetic resin and glass slides enable HaloTag mediated protein immobilization and display. Further development of new HaloTag surfaces, fluorescent ligands and ligands with other functionalities should allow creation of additional applications of HaloTag. The availability of HaloTag reactive intermediate substrates (reactive ligands), consisting essentially of the binding group carrying a reactive group, enables creation of custom reagents by adding any functional group of interest following standard chemical synthesis. This substrate has allowed the development of novel applications by other users [24
]. Several key applications of HaloTag are described below.