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.
Highlights
The biarsenical-tetracysteine tag was initially conceived of and developed for site-specific small-molecule labeling of proteins both in vitro and in living cells [1,2], to overcome the limitations resulting from the fairly large size of genetically encoded fluorescent proteins such as eGFP (27 kDa) [3].This method relies on the high affinity binding of an arsenic-containing fluorescein derivative, termed fluorescein arsenical hairpin binder (FlAsH), and a peptide bearing four cysteine residues with the general structure CCXXCC
We have explored the use of different linkers and TC sequences with the aim of improving the protein immobilization conditions
CrAsH (1 and 2) or carboxyfluorescein (3 and 4) derivatives followed by a blocking step with ethanolamine [16]
Summary
The biarsenical-tetracysteine tag was initially conceived of and developed for site-specific small-molecule labeling of proteins both in vitro and in living cells [1,2], to overcome the limitations resulting from the fairly large size of genetically encoded fluorescent proteins such as eGFP (27 kDa) [3]. This method relies on the high affinity binding of an arsenic-containing fluorescein derivative, termed fluorescein arsenical hairpin binder (FlAsH), and a peptide bearing four cysteine residues with the general structure CCXXCC. The developed technology has been applied to the direct immobilization of recombinant proteins via on-chip ligation of protein thioesters with excellent intensities and short reaction times
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