Visualizing and Manipulating Biological Processes by Using HaloTag and SNAP‐Tag Technologies

Abstract

Visualizing and manipulating behavior of proteins is crucial to understanding the physiology of the cell. Methods of biorthogonal protein labelling are important tools to attain this goal. In this review, we discuss advances in probe technology specific for self-labelling protein tags, focusing mainly on the application of HaloTag and SNAP-tag systems. We describe the latest developments in small molecule probes that enable fluorogenic (no wash) imaging and super-resolution fluorescence microscopy. In addition, we cover several methodologies that enable the perturbation or manipulation of protein behavior and function towards the control of biological pathways. Thus, the current technical advancement on the HaloTag and SNAP-tag systems are becoming powerful tools to enable the visualization and manipulating of biological processes, providing invaluable scientific insights that are otherwise difficult to obtain using traditional methodologies. As the multiplex self-labelling protein tag systems continue to be developed and expanded, the utility of these protein tags will allow researchers to address previously inaccessible questions at the forefront of biology.

Fluorescent proteins of the EosFP clade: intriguing marker tools with multiple photoactivation modes for advanced microscopy

“…Therefore, we envision that there are still considerable opportunities for advances in FP marker technology. Likewise, we expect progress with other genetically encoded labeling techniques not covered in this review, e.g., chemical tags such as Halo or SNAP for protein labeling 161 or light-up aptamers for RNA labeling. [162][163][164] These approaches employ synthetic dyes with excellent fluorescence properties, superior to those of FP-based labels, but require an external supply of fluorophores, limiting their versatility and ease of use.…”

Transient Fluorescence Labeling: Low Affinity—High Benefits

“…Existing low-affinity labeling methods are compatible with different microscopy setups, ranging from common wide-field and TIRF microscopy to lattice light-sheet microscopy [124]. 1 A combination of genetically encoded part with organic fluorogens added externally; 2 includes other methods, based on probe that transiently interacts with a specific target protein; 3 could be used either with organic dyes or fluorescent proteins; 4 and LIVE-PAINT.…”

“…These tags are convenient but have a limited color palette. Finally, a diverse set of exogenously applied chemical dyes can be permanently attached to the protein of interest fused with specially designed genetically encoded enzymatic tags, such as SNAP-tag and HaloTag (reviewed in [4]).…”

Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling

“…Since its development and commercialization by Promega, HaloTag® has become a valuable research tool for a broad range of applications (Figure 1A) including protein purification 5 and immobilization 6 , enhancement of the soluble expression of recombinant proteins 7 , cellular protein imaging 8,9 , imaging in vivo 10 , and single-molecule studies [11][12][13] . The technology is applicable for analyses of protein-protein and protein-nucleic acid interactions 14,15 , proteome stress 16,17 , protein folding and aggregation 18,19 , dynamics and hydration [20][21][22] , or cellpermeability 23 .…”