Snap-Tag

A snap- tag is a protein tag, which makes it possible with a fluorescent dye to label a protein in cells of a specific cell culture with a high quantum yield.

Properties

Standard studies are nowadays conducted with protein tags, for example, fluorescent proteins such as GFP ( Green Fluorescent Protein ) or YFP (Yellow fluorescent protein). The methods are indeed relatively easy to perform, because the underlying techniques are now well established ( formation of fusion proteins and specific expression in living cells ); on the other hand, the photo-physical properties, the proteins are generally not suitable in order to operate the single-molecule spectroscopy. They have in comparison to the commercially available dyes in a very much lower fluorescence quantum yield and can be rapidly destroyed by excitation with a focused laser beam in the course of a photo- bleaching in the rule.

The so-called SNAP- protein is a derivative of a ubiquitous enzyme in mammalian cells, the O -6- Alkylguaninalkyltransferase (AGT ) which is usually in the organism with the task of repairing defects in DNA - guanosines. A similar function has the snap- protein in the course of the labeling of the fusion protein.

Need to mark, as with all protein tags, the first DNA sequence of the partial fragment of AGT are an in frame compliant inserted into the DNA sequence of the protein to be labeled. In addition, any fluorescence dye to be chemically coupled to the SNAP- substrate BG -NH2. Via an amino group on the substrate of the NHS ester of the dye to be used is bound by a SN2 -covalently to the substrate. Subsequently, the substrate with the dye must now diffuse through the membrane into a cell in cell culture. Is it there to the snap- protein, the ether contained in the substrate is cleaved by the enzymatic component of the AGT fusion protein and a guanine is released by direct covalent bond between the protein to be examined, and the dye is formed.

Reaction scheme

References and further reading

  • Keppler, A. et al. (2004): Labeling of fusion proteins of O6- alkylguanine - DNA alkyltransferase with small molecules in vivo and in vitro. In: Methods. Vol 32, pp. 437-444. PMID 15003606
  • Keppler, A. et al. (2004): Labeling of fusion proteins with synthetic fluorophores in live cells. In: Proc. Natl. Acad. Sci. U.S.A. Vol 101, pp. 9955-9959. PMID 15226507
  • Juillerat, A. et al. (2005 ): Engineering substrates specificity of O6- alkylguanine - DNA alkyltransferase for specific protein labeling in living cells. In: ChemBioChem. Vol 6, pp. 1263-1269. PMID 15934048
  • Brecht, A. & Gibbs, T. ( 2005): self-labeling protein tags. In: Bioforum. Born 2005, No. 6, pp. 50-51.
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