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Investigation of GST fusion proteins for screening protein interactions
Author(s) -
Thomsen Kristen,
Barnett Jason,
Elder Darcie,
Caldwell Benjamin D,
Ducey Michael W
Publication year - 2008
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.22.1_supplement.1057.8
Subject(s) - fusion protein , fluorescence anisotropy , chemistry , glutathione , biochemistry , fluorescence , protein–protein interaction , cleavage (geology) , bimolecular fluorescence complementation , biophysics , recombinant dna , enzyme , biology , gene , paleontology , physics , quantum mechanics , membrane , fracture (geology)
Proteins of interest are commonly expressed as fusion protein conjugates with Glutathione S‐Transferase (GST) due to the relative ease with which GST can be purified. Expression vectors may have proteolytic cleavage sites engineered into the proteins sequence so that the protein of interest may be separated from GST and isolated for further study; however, these enzymes can be expensive and cleavage may be incomplete, reducing the usable yield. We investigated fluorescence anisotropy as a method for examining the interaction of GST fusion proteins. Fluorescence studies often use ligands specific for the protein of interest; however some proteins may not have natural probes suitable for fluorescence studies. We investigated the possibility of measuring the interaction of GST fusion proteins via glutathione (Glu‐Cys‐Gly, or GSH) labeled with a fluorecein‐maleimide. Titration with GST demonstrated an increase in fluorescence anisotropy, indicating GSH‐Fl ligand binding. Results suggest that fluorescence anisotropy of GST‐fusion protein interaction could be a preliminary or complementary screening method for examining protein interactions, especially those whose natural ligands are not spectroscopically observable.