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Molecular Interaction Assays Based On A Self Labeling Protein Tag
Author(s) -
Gedge L.,
Muentener K.,
Gibbs T.,
Brecht A.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a961-b
Subject(s) - fluorophore , fkbp , chemistry , biotinylation , streptavidin , protein–protein interaction , target protein , förster resonance energy transfer , protein tag , covalent bond , immunoprecipitation , biochemistry , recombinant dna , fluorescence , fusion protein , green fluorescent protein , biophysics , biotin , microbiology and biotechnology , biology , physics , organic chemistry , quantum mechanics , gene
Protein‐tags are widely used for purification and detection of recombinant proteins. Recently a self‐labeling protein tag, the SNAP‐tag, has been introduced. This tag reacts covalently with a wide range of substrates that are based on benzylguanine (BG). These substrates can be fluorescent labels, affinity labels, or even modified surfaces. The utility of this covalently self‐modifying protein tag will be discussed using 3 example applications based on the protein‐protein interaction system FKBP‐FRB. Both proteins were expressed as SNAP‐tag fusions and used in the following assays: 1) A microplate interaction assay based on SNAP‐FKBP labelled with BG‐biotin and bound to a streptavidin coated microplate and SNAP‐FRB labelled with a BG‐fluorophore. The interaction showed the expected clear dependence on the drug Rapamycin. 2) A homogeneous phase interaction assay based on a donor‐acceptor pair for time‐resolved FRET. The FKBP‐FRB interaction worked with an affinity consistent with literature values and the effect of inhibitors of the interaction was found as predicted. 3) A pull‐down assay using SNAP‐FKBP immobilized to a Sepharose resin modified with BG and SNAP‐FRB labelled with a BG‐fluorophore. Specific pull down of FRB could be shown in the presence of Rapamycin. The system worked without any significant difference in purified samples or in raw bacterial lysates. In summary, the data demonstrates a broad potential for covalently self‐labeling protein tags. This work was in part funded by a CTI‐grant from the Swiss government (CTI‐grant 6255).