Flash labeling of a nuclear receptor domain (D domain of ultraspiracle) fused to tetracysteine tag.
Biarsenical fluorescein compounds feature unique fluorescence characteristics and special binding mechanism to tetracysteine tags with certain structures and these dyes offer a feasible method for site specific labeling of heterologously expressed proteins. We aimed FlAsH fluorescent labeling of tetracysteine fused hinge region of the ultraspiracle from Drosophila melanogaster (DmUSP-D domain) to facilitate functional studies of this receptor domain. A CCPGCC tetracysteine motif was integrated between His6, Gateway attB1, and Flag tags and attached to the N-terminus of the DmUSP-D. The fusion protein was expressed in Esherichia coli and the FlAsH labeling was performed in bacterial extracts, under conditions which are compatible with receptor function. The dye was bound to the tetracysteine tag with high affinity and complex stability and the labeling proved to be specific for the target fusion protein. Results indicate that FlAsH labeling of the internal CCPGCC motif can be a valuable tool for the functional characterisation of any nuclear receptor domains.
Szécsi
M
M
Department of General Zoology and Endocrinology, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany. szecsim@endoc.szote.u-szeged.hu
Spindler-Barth
Margarethe
M
D000595:Amino Acid Sequence; D000818:Animals; D001152:Arsenicals / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001152:Arsenicals; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C066030:USP protein, Drosophila; D003545:Cysteine