title: Specific covalent immobilization of proteins through dityrosine cross-links.
abstract: Dityrosine cross-links are widely observed in nature in structural proteins such as elastin and silk. Natural oxidative cross-linking between tyrosine residues is catalyzed by a diverse group of metalloenzymes. Dityrosine formation is also catalyzed in vitro by metal-peptide complexes such as Gly-Gly-His-Ni(II). On the basis of these observations, a system was developed to specifically and covalently surface immobilize proteins through dityrosine cross-links. Methacrylate monomers of the catalytic peptide Gly-Gly-His-Tyr-OH (GGHY) and the Ni(II)-chelating group nitrilotriacetic acid (NTA) were copolymerized with acrylamide into microbeads. Green fluorescent protein (GFP), as a model protein, was genetically tagged with a tyrosine-modified His6 peptide on its carboxy terminus. GFP-YGH6, specifically associated with the NTA-Ni(II) groups, was covalently coupled to the bead surface through dityrosine bond formation catalyzed by the colocalized GGHY-Ni(II) complex. After extensive washing with EDTA to disrupt metal coordination bonds, we observed that up to 75% of the initially bound GFP-YGH6 remained covalently bound to the bead while retaining its structure and activity. Dityrosine cross-linking was confirmed by quenching the reaction with free tyrosine. The method may find particular utility in the construction and optimization of protein microarrays.
authors.affiliation: Department of Bioengineering, University of Utah, 20 South 2030 East, Room 506, Salt Lake City, UT 84112, USA.
authors.forename: Betsy J
authors.initials: BJ
authors.lastname: Endrizzi
authors.forename: Gang
authors.initials: G
authors.lastname: Huang
authors.forename: Patrick F
authors.initials: PF
authors.lastname: Kiser
authors.forename: Russell J
authors.initials: RJ
authors.lastname: Stewart
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry*; D008863:Microspheres; D009532:Nickel / Q000737:chemistry*; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*; D040081:Protein Array Analysis / Q000379:methods; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D010455:Peptides; D014443:Tyrosine; D009532:Nickel; C007543:dityrosine