title: Site-specific, covalent attachment of proteins to a solid surface.
abstract: Immobilized and site-specifically labeled proteins are becoming invaluable tools in proteomics. Here, we describe a strategy to attach a desired protein to a solid surface in a covalent, site-specific manner. This approach employs an enzymatic posttranslational modification method to site-specifically label a target protein with an azide; an alternative substrate for protein farnesyl transferase containing an azide group was developed for this purpose. A bio-orthogonal Cu(I)-catalyzed cycloaddition reaction is then used to covalently attach the protein to agarose beads bearing an alkyne functional group. We demonstrate that both the azide incorporation and the capture steps can be performed on either a purified protein target or on a protein present within a complex mixture. This approach involves the use of a four-residue tag which is significantly smaller than most other tags reported to date and results in covalent immobilization of the target protein. Hence it should have significant applicability in protein science.
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Duckworth
authors.forename: Juhua
authors.initials: J
authors.lastname: Xu
authors.forename: T Andrew
authors.initials: TA
authors.lastname: Taton
authors.forename: Athena
authors.initials: A
authors.lastname: Guo
authors.forename: Mark D
authors.initials: MD
authors.lastname: Distefano
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D017931:DNA Primers; D009682:Magnetic Resonance Spectroscopy; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D017931:DNA Primers; D011506:Proteins