title: pGSTag--a versatile bacterial expression plasmid for enzymatic labeling of recombinant proteins.
abstract: We report on the construction of a plasmid, pGSTag, that directs the expression in E. coli of a glutathione S-transferase fusion protein that contains a high affinity phosphorylation site by protein kinase-A (PK-A). The fusion protein, following purification from crude bacterial lysates by substrate affinity chromatography, can be labeled in vitro to high specific activity with purified PK-A and 32P-gamma-ATP. Because labeling takes place while the fusion protein is immobilized on a solid support, the unincorporated label and enzyme can be washed away. Using the leucine-zipper domains of cAMP response element binding (CREB) proteins and CCAAT/enhancer binding protein (C/EBP)-like proteins as a model system, we show that the labeled protein, after elution from the affinity resin, can be used as a probe to detect interacting (dimerizing) species in a nitrocellulose-based ligand blot assay. The utility of this system for the creation of labeled protein probes is discussed.
authors: Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston 02114.
authors: D
authors: D
authors: Ron
authors: H
authors: H
authors: Dressler
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010957:Plasmids; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D011494:Protein Kinases