SNAP-tag® is a self-labeling protein tag commercially available in various expression vectors. SNAP-tag is a 182 residues polypeptide (19.4 kDa) that can be fused to any protein of interest and further specifically and covalently tagged with a suitable ligand, such as a

fluorescent dye A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...

. Since its introduction, SNAP-tag has found numerous applications in biochemistry and for the investigation of the function and localisation of proteins and

enzymes Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different molecule ...

in living cells. Compared to the current standard labelling methods used in fluorescence microscopy, the use of SNAP-tag presents significant advantages. SNAP-tag® is a registered trademark of New England Biolabs, Inc.

Applications

Cell biology utilizes tools that allow manipulation and visualization of proteins in living cells. An important example is the use of fluorescent proteins, such as the

green fluorescent protein The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish ''Aequorea ...

(GFP) or yellow fluorescent protein (YFP). Molecular biology methods allow these fluorescent proteins to be introduced and expressed in living cells as fusion proteins. However, the photo-physical properties of the fluorescent proteins are generally not suited for single-molecule spectroscopy. Fluorescent proteins have, in comparison to commercially available dyes, a much lower fluorescence quantum yield and are quickly destroyed upon excitation with a focused laser beam (photobleaching). The SNAP-tag® protein is an engineered version of the ubiquitous mammalian enzyme AGT, encoded in humans by the O-6-methylguanine-DNA methyltransferase (MGMT) gene. SNAP-tag was obtained using a directed evolution strategy, leading to a hAGT variant that accepts ''O''⁶-benzylguanine derivatives instead of repairing alkylated guanine derivatives in damaged DNA. An orthogonal tag, called CLIP-tag™, was further engineered from SNAP-tag to accept ''O''²-benzylcytosine derivatives as substrates, instead of ''O''⁶-benzylguanine. Therefore, Clip-tag- and SNAP-tag-fused proteins can be labeled simultaneously in the same cells. A split-SNAP-tag version suitable for protein complementation assay and protein-protein interaction studies was later developed. Apart from fluorescence microscopy, SNAP-tag and CLIP-tag have proven useful in the elucidation of numerous biological processes, including the identification of multiprotein complexes using various approaches such as

FRET A fret is any of the thin strips of material, usually metal wire, inserted laterally at specific positions along the neck or fretboard of a stringed instrument. Frets usually extend across the full width of the neck. On some historical instrume ...

, cross-linking, proximity ligation assay, as well as the purification of insulin secretory granules of distinct age by doing pulse-chase experiments Other application include the measurement of protein half-lives in vivo, and small molecule-protein interactions. SNAP-tag® is a registered trademark of New England Biolabs, Inc. CLIP-tag™ is a trademark of New England Biolabs, Inc.

References

External links

* Darstellung SNAP-Tag und CLIP-Tag (NEB) * ''Self Labeling Protein Tags.'' In: ''Bioforum.'' Jg. 2005, Nr. 6, S. 50-51. Biochemistry detection methods

Applications

See also

References

Further reading

External links