title: High Affinity Immobilization of Proteins Using the CrAsH/TC Tag.
abstract: Protein microarrays represent important tools for biomedical analysis. We have recently described the use of the biarsenical-tetracysteine (TC) tag for the preparation of protein microarrays. The unique feature of this tag enables the site-specific immobilization of TC-containing proteins on biarsenical-modified surfaces, resulting in a fluorescence enhancement that allows the direct quantification of the immobilized proteins. Moreover, the reversibility of the binding upon incubation with large quantities of thiols permits the detachment of the proteins from the surface, thereby enabling recovery of the substrate to extend the life time of the slide. Herein, we describe our recent results that further extend the applicability of the CrAsH/TC tag to the fabrication of biochips. With this aim, the immobilization of proteins on surfaces has been investigated using two different spacers and two TC tags, the minimal TC sequence (CCPGCC) and an optimized motif (FLNCCPGCCMEP). While the minimal peptide motif enables a rapid recycling of the slide, the optimized TC sequence reveals an increased affinity due to its greater resistance to displacement by thiols. Moreover, the developed methodology was applied to the immobilization of proteins via on-chip ligation of recombinant protein thioesters.
authors.affiliation: Department of Chemical Biology, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany. Janine.Schulte-Zweckel@mpi-dortmund.mpg.de.
authors.forename: Janine
authors.initials: J
authors.lastname: Schulte-Zweckel
authors.affiliation: Department of Chemical Biology, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany. federicarosi80@gmail.com.
authors.forename: Federica
authors.initials: F
authors.lastname: Rosi
authors.affiliation: Department of Chemical Biology, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany. sreenu.d@intogenbio.com.
authors.forename: Domalapally
authors.initials: D
authors.lastname: Sreenu
authors.affiliation: Chimera Biotec GmbH, Emil-Figge-Str., 76 A, D-44227 Dortmund, Germany. schroeder@chimera-biotec.com.
authors.forename: Hendrik
authors.initials: H
authors.lastname: Schröder
authors.affiliation: Institute of Biological Interfaces (IBG1), Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz Platz, D-76344 Eggenstein-Leopoldshafen, Germany. niemeyer@kit.edu.
authors.forename: Christof M
authors.initials: CM
authors.lastname: Niemeyer
authors.affiliation: Department of Biomedicinal Chemistry, Institute of Advanced Chemistry of Catalonia, Jordi Girona 18-26, 08034 Barcelona, Spain. gemma.triola@iqac.csic.es.
authors.forename: Gemma
authors.initials: G
authors.lastname: Triola
mesh_terms: D003545:Cysteine / Q000737:chemistry; D055767:Immobilized Proteins / Q000737:chemistry*; D009942:Organometallic Compounds / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D040081:Protein Array Analysis / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: C518706:CrAsH compound; D055767:Immobilized Proteins; D009942:Organometallic Compounds; D010455:Peptides; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D003545:Cysteine
keywords: CrAsH; FlAsH; on-chip ligation; protein immobilization; protein microarray; site-selective