Cargando…
High Affinity Immobilization of Proteins Using the CrAsH/TC Tag
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 biarsen...
Autores principales: | , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2016
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6273389/ https://www.ncbi.nlm.nih.gov/pubmed/27338319 http://dx.doi.org/10.3390/molecules21060750 |
_version_ | 1783377373281910784 |
---|---|
author | Schulte-Zweckel, Janine Rosi, Federica Sreenu, Domalapally Schröder, Hendrik Niemeyer, Christof M. Triola, Gemma |
author_facet | Schulte-Zweckel, Janine Rosi, Federica Sreenu, Domalapally Schröder, Hendrik Niemeyer, Christof M. Triola, Gemma |
author_sort | Schulte-Zweckel, Janine |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-6273389 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-62733892018-12-28 High Affinity Immobilization of Proteins Using the CrAsH/TC Tag Schulte-Zweckel, Janine Rosi, Federica Sreenu, Domalapally Schröder, Hendrik Niemeyer, Christof M. Triola, Gemma Molecules Article 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. MDPI 2016-06-08 /pmc/articles/PMC6273389/ /pubmed/27338319 http://dx.doi.org/10.3390/molecules21060750 Text en © 2016 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Schulte-Zweckel, Janine Rosi, Federica Sreenu, Domalapally Schröder, Hendrik Niemeyer, Christof M. Triola, Gemma High Affinity Immobilization of Proteins Using the CrAsH/TC Tag |
title | High Affinity Immobilization of Proteins Using the CrAsH/TC Tag |
title_full | High Affinity Immobilization of Proteins Using the CrAsH/TC Tag |
title_fullStr | High Affinity Immobilization of Proteins Using the CrAsH/TC Tag |
title_full_unstemmed | High Affinity Immobilization of Proteins Using the CrAsH/TC Tag |
title_short | High Affinity Immobilization of Proteins Using the CrAsH/TC Tag |
title_sort | high affinity immobilization of proteins using the crash/tc tag |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6273389/ https://www.ncbi.nlm.nih.gov/pubmed/27338319 http://dx.doi.org/10.3390/molecules21060750 |
work_keys_str_mv | AT schultezweckeljanine highaffinityimmobilizationofproteinsusingthecrashtctag AT rosifederica highaffinityimmobilizationofproteinsusingthecrashtctag AT sreenudomalapally highaffinityimmobilizationofproteinsusingthecrashtctag AT schroderhendrik highaffinityimmobilizationofproteinsusingthecrashtctag AT niemeyerchristofm highaffinityimmobilizationofproteinsusingthecrashtctag AT triolagemma highaffinityimmobilizationofproteinsusingthecrashtctag |