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Molecules 2016, 21(6), 750; doi:10.3390/molecules21060750

High Affinity Immobilization of Proteins Using the CrAsH/TC Tag

1
Department of Chemical Biology, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany
2
Chimera Biotec GmbH, Emil-Figge-Str., 76 A, D-44227 Dortmund, Germany
3
Institute of Biological Interfaces (IBG1), Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz Platz, D-76344 Eggenstein-Leopoldshafen, Germany
4
Department of Biomedicinal Chemistry, Institute of Advanced Chemistry of Catalonia, Jordi Girona 18-26, 08034 Barcelona, Spain
*
Author to whom correspondence should be addressed.
Academic Editor: Chris Frost
Received: 5 May 2016 / Revised: 2 June 2016 / Accepted: 3 June 2016 / Published: 8 June 2016
(This article belongs to the Special Issue Biomolecules Modification)
View Full-Text   |   Download PDF [1495 KB, uploaded 8 June 2016]   |  

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. View Full-Text
Keywords: protein microarray; protein immobilization; FlAsH; CrAsH; site-selective; on-chip ligation protein microarray; protein immobilization; FlAsH; CrAsH; site-selective; on-chip ligation
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Schulte-Zweckel, J.; Rosi, F.; Sreenu, D.; Schröder, H.; Niemeyer, C.M.; Triola, G. High Affinity Immobilization of Proteins Using the CrAsH/TC Tag. Molecules 2016, 21, 750.

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