title: Site-specific and covalent attachment of his-tagged proteins by chelation assisted photoimmobilization: a strategy for microarraying of protein ligands.
abstract: A novel strategy for site-specific and covalent attachment of proteins has been developed, intended for robust and controllable immobilization of histidine (His)-tagged ligands in protein microarrays. The method is termed chelation assisted photoimmobilization (CAP) and was demonstrated using human IgG-Fc modified with C-terminal hexahistidines (His-IgGFc) as the ligand and protein A as the analyte. Alkanethiols terminated with either nitrilotriacetic acid (NTA), benzophenone (BP), or oligo(ethylene glycol) were synthesized and mixed self-assembled monolayers (SAMs) were prepared on gold and thoroughly characterized by infrared reflection absorption spectroscopy (IRAS), ellipsometry, and contact angle goniometry. In the process of CAP, NTA chelates Ni(2+) and the complex coordinates the His-tagged ligand in an oriented assembly. The ligand is then photoimmobilized via BP, which forms covalent bonds upon UV light activation. In the development of affinity biosensors and protein microarrays, site-specific attachment of ligands in a fashion where analyte binding sites are available is often preferred to random coupling. Analyte binding performance of ligands immobilized either by CAP or by standard amine coupling was characterized by surface plasmon resonance in combination with IRAS. The relative analyte response with randomly coupled ligand was 2.5 times higher than when site-specific attachment was used. This is a reminder that also when immobilizing ligands via residues far from the binding site, there are many other factors influencing availability and activity. Still, CAP provides a valuable expansion of protein immobilization techniques since it offers attractive microarraying possibilities amenable to applications within proteomics.
authors.affiliation: Division of Molecular Physics, ‡Division of Organic Chemistry, and §Division of Applied Physics; Department of Physics, Chemistry and Biology, Linköping University , SE-581 83 Linköping, Sweden.
authors.forename: Emma M
authors.initials: EM
authors.lastname: Ericsson
authors.forename: Karin
authors.initials: K
authors.lastname: Enander
authors.forename: Lan
authors.initials: L
authors.lastname: Bui
authors.forename: Ingemar
authors.initials: I
authors.lastname: Lundström
authors.forename: Peter
authors.initials: P
authors.lastname: Konradsson
authors.forename: Bo
authors.initials: B
authors.lastname: Liedberg
mesh_terms: D002614:Chelating Agents / Q000737:chemistry*; D006639:Histidine / Q000031:analogs & derivatives / Q000737:chemistry*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D008024:Ligands; D015394:Molecular Structure; D055668:Photochemical Processes; D040081:Protein Array Analysis
chemical_list: D002614:Chelating Agents; D007141:Immunoglobulin Fc Fragments; D008024:Ligands; D006639:Histidine