SNAP-tag® is a self-labeling protein tag commercially available in various expression vectors. SNAP-tag is a 182 residue 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. Since its introduction, SNAP-tag has found numerous applications in biochemistry and for the investigation of the function and localisation of proteins and enzymes in living cells.[1]
Applications
editCell 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 (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,[2] encoded in humans by the O-6-methylguanine-DNA methyltransferase (MGMT) gene. SNAP-tag was obtained using a directed evolution strategy,[3] leading to a hAGT variant that accepts O6-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 O2-benzylcytosine derivatives as substrates, instead of O6-benzylguanine.[4] 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.[5]
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,[6] cross-linking,[6] proximity ligation assay,[7] as well as the purification of insulin secretory granules of distinct age by doing pulse-chase experiments [8] Other application include the measurement of protein half-lives in vivo,[9] and small molecule-protein interactions.[10] SNAP-tag® is a registered trademark of New England Biolabs, Inc. CLIP-tag™ is a trademark of New England Biolabs, Inc.
See also
editReferences
edit- ^ Crivat G; Taraska JW (January 2012). "Imaging proteins inside cells with fluorescent tags". Trends in Biotechnology. 30 (1): 8–16. doi:10.1016/j.tibtech.2011.08.002. PMC 3246539. PMID 21924508.
- ^ Juillerat A; Gronemeyer T; Keppler A; Gendreizig S; Pick H; Vogel H; Johnsson K (April 2003). "Directed evolution of O6-alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo". Chemistry and Biology. 10 (4): 313–317. doi:10.1016/S1074-5521(03)00068-1. PMID 12725859.
- ^ Mollwitz, Birgit; Brunk, Elizabeth; Schmitt, Simone; Pojer, Florence; Bannwarth, Michael; Schiltz, Marc; Rothlisberger, Ursula; Johnsson, Kai (2012-02-07). "Directed Evolution of the Suicide Protein O6-Alkylguanine-DNA Alkyltransferase for Increased Reactivity Results in an Alkylated Protein with Exceptional Stability". Biochemistry. 51 (5): 986–994. doi:10.1021/bi2016537. ISSN 0006-2960. PMID 22280500.
- ^ Gautier A; Juillerat A; Heinis C; Corrêa IR Jr; Kindermann M; Beaufils F; Johnsson K (February 2008). "An engineered protein tag for multiprotein labeling in living cells". Chemistry and Biology. 15 (2): 128–136. doi:10.1016/j.chembiol.2008.01.007. PMID 18291317.
- ^ Mie M; Naoki T; Uchida K; Kobatake E (October 2012). "Development of a split SNAP-tag protein complementation assay for visualization of protein-protein interactions in living cells". Analyst. 137 (20): 4760–4765. Bibcode:2012Ana...137.4760M. doi:10.1039/c2an35762c. PMID 22910969. S2CID 6217675.
- ^ a b Maurel D; Comps-Agrar L; Brock C; Rives ML; Bourrier E; Ayoub MA; Bazin H; Tinel N; Durroux T; Prézeau L; Trinquet E; Pin JP (June 2008). "Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization". Nature Methods. 5 (6): 561–567. doi:10.1038/nmeth.1213. PMC 2642604. PMID 18488035.
- ^ Gu GJ; Friedman M; Jost C; Johnsson K; Kamali-Moghaddam M; Plückthun A; Landegren U; Söderberg O (January 2013). "Protein tag-mediated conjugation of oligonucleotides to recombinant affinity binders for proximity ligation". New Biotechnology. 30 (2): 144–152. doi:10.1016/j.nbt.2012.05.005. PMID 22664266.
- ^ Neukam, Martin; Ganß, Katharina; Vasiljević, Jovana; Broichhagen, Johannes; Johnsson, Kai; Kurth, Thomas; Solimena, Michele (2020-06-03). "Purification of age-distinct insulin secretory granules through antigen restriction using the CLIP-tag": 2020.06.03.103770. doi:10.1101/2020.06.03.103770. S2CID 219535633.
{{cite journal}}: Cite journal requires|journal=(help) - ^ Bodor DL; Rodríguez MG; Moreno N; Jansen LE (June 2012). Analysis of protein turnover by quantitative SNAP-based pulse-chase imaging. Vol. 55. pp. Unit8.8. doi:10.1002/0471143030.cb0808s55. hdl:10400.7/614. ISBN 978-0471143031. PMID 23129118.
{{cite book}}:|journal=ignored (help) - ^ Chidley C; Haruki H; Pedersen MG; Muller E; Johnsson K (June 2011). "A yeast-based screen reveals that sulfasalazine inhibits tetrahydrobiopterin biosynthesis". Nature Chemical Biology. 7 (6): 375–383. doi:10.1038/nchembio.557. PMID 21499265.
Further reading
edit- Keppler A; Kindermann M; Gendreizig S; Pick H; Vogel H; Johnsson K (2004). "Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro". Methods. 32 (4): 437–444. doi:10.1016/j.ymeth.2003.10.007. PMID 15003606.
- Keppler A; Pick H; Arrivoli C; Vogel H; Johnsson K (2004). "Labeling of fusion proteins with synthetic fluorophores in live cells". Proc. Natl. Acad. Sci. U.S.A. 101 (27): 9955–9959. Bibcode:2004PNAS..101.9955K. doi:10.1073/pnas.0401923101. PMC 454197. PMID 15226507.
- Juillerat A; Heinis C; Sielaff I; Barnikow J; Jaccard H; Kunz B; Terskikh A; Johnsson K (2005). "Engineering substrate specificity of O6-alkylguanine-DNA alkyltransferase for specific protein labeling in living cells". ChemBioChem. 6 (7): 1263–1269. doi:10.1002/cbic.200400431. PMID 15934048. S2CID 23926764.
External links
edit- Darstellung SNAP-Tag und CLIP-Tag (NEB)
- Self Labeling Protein Tags. In: Bioforum. Jg. 2005, Nr. 6, S. 50-51.