title: Affinity tag for protein purification and detection based on the disulfide-linked complex of InaD and NorpA.
abstract: Affinity tags are not only used for the expression and purification of recombinant proteins but also for the detection of protein-protein interactions. Common problems with many affinity tags are excessive length, which may interfere with the structure and function of tagged proteins, and low affinity and/or specificity for primary detection and purification agents. Preliminary results suggest that the C-terminalfive residues of the Drosophila protein NorpA, based on the short, covalent interaction they make with the N-terminal PDZ domain (PDZI) of InaD, are useful as a general affinity tag. First, a PDZI-alkaline phosphatase fusion protein specifically detects both its physiological ligand and a heterologous protein expressing the NorpA C-terminal five residues. The interaction of PDZI with a NorpA-tagged protein is reversible by a reducing agent, which allows nitrocellulose membranes to be stripped completely and reused. In addition, a NorpA-tagged protein can specifically bind to immobilized PDZI resin, while other cellular proteins are washed through. After washing, the NorpA-tagged protein is eluted by a reducing buffer. The NorpA tag's short length makes it the smallest affinity tag available, and its specific and high-affinity interaction with PDZI could yield a powerful system that improves on currently available technology.
authors.affiliation: The University of North Carolina at Chapel Hill, 27599-7365, USA.
authors.forename: Michelle E
authors.initials: ME
authors.lastname: Kimple
authors.forename: John
authors.initials: J
authors.lastname: Sondek
mesh_terms: D000345:Affinity Labels; D000818:Animals; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry*; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D005136:Eye Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D046911:Macromolecular Substances; D015336:Molecular Probe Techniques; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054799:Phospholipase C beta; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D010738:Type C Phospholipases / Q000737:chemistry*
chemical_list: D000345:Affinity Labels; D004220:Disulfides; D029721:Drosophila Proteins; D005136:Eye Proteins; D046911:Macromolecular Substances; D011506:Proteins; C091568:inaD protein, Drosophila; D010738:Type C Phospholipases; C468371:NorpA protein, Drosophila; D054799:Phospholipase C beta; D043265:Phosphatidylinositol Diacylglycerol-Lyase