Abstract
The knowledge of interactions among functional proteins helps researchers understand disease mechanisms and design potential strategies for treatment. As a general approach, the fluorescent and affinity tags were employed for exploring this field by labeling the Protein of Interest (POI). However, the autofluorescence and weak binding strength significantly reduce the accuracy and specificity of these tags. Conversely, HaloTag, a novel self-labeling enzyme (SLE) tag, could quickly form a covalent bond with its ligand, enabling fast and specific labeling of POI. These desirable features greatly increase the accuracy and specificity, making the HaloTag a valuable system for various applications ranging from imaging to immobilization of POI. Notably, the HaloTag technique has already been successfully employed in a series of studies with excellent efficiency. In this review, we summarize the development of HaloTag and recent advanced investigations associated with HaloTag, including in vitro imaging (e.g., POI imaging, cellular condition monitoring, microorganism imaging, system development), in vivo imaging, biomolecule immobilization (e.g., POI collection, protein/nuclear acid interaction and protein structure analysis), targeted degradation (e.g., L-AdPROM), and more. We also present a systematic discussion regarding the future direction and challenges of the HaloTag technique.
Highlights
A series of biological processes are kept happening in the body, which was triggered by complex interactions between biomacromolecule
There are several challenges related to the HaloTag system, of which researchers should be attentive. (a) the modification will improve its solubility, the structure of HaloTag ligand (HTL) may still affect its circulation, especially for in vivo applications; in addition to innovative designs of HTLs, the pre-loading strategy (e.g., HaloTag-protein of interest (POI) labeled with HTL in vitro before injection) may act as a potential solution; in contrast, long-term imaging could be achieved via multiple administration of imaging HTL once high solubility was achieved. (b) Most HTLs used in studies are self-synthesized according to the purpose of their use
The 1:1 ratio labeling capacity greatly ensures the reproduction of complex of HaloTag-fused protein and HTLs, such as PET tracer (i.e., HaloTag fused nanobody and radioactive isotope-labeled HTLs), well meeting the requirements of clinical application
Summary
A series of biological processes (e.g., development of diseases) are kept happening in the body, which was triggered by complex interactions between biomacromolecule (e.g., protein-protein or protein-nucleic acid). The impurity of harvested proteins could be caused by unspecific binding (e.g., His-tag) or a decrease in the brightness of fluorescence, etc These disadvantages potentially restrain their applications in studies that require high accuracy (e.g., miRNA-protein interaction or POI tracking studies). Three prominent examples are the CLIP-tag/ SNAP-tag (19.4 kDa), ACPtag (9 kDa) and HaloTag (33 kDa) (Table 1) These SLEs share several features, including relatively small size, high stability, and fast-reacting kinetics, allowing fused POI to be labeled by tag-specific substrates with extremely high specificity and efficiency. We summarize the developments of HaloTag technology, in particular, recent applications of HaloTag for POI tracking (e.g., in vitro and in vivo imaging), immobilization (e.g., protein-reacting mRNA analysis and protein structure study), targeted degradation (e.g., chloroalkane-containing proteolysis targeting chimeric (HaloPROTACs)) and other applications (e.g., differentiation of stem cell), is highlighted (Scheme 1). Prospects and challenges for future developments of HaloTag are systemically discussed
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