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Off‐Pathway‐Sensitive Protein‐Splicing Screening Based on a Toxin/Antitoxin System
Protein‐splicing domains are frequently used engineering tools that find application in the in vivo and in vitro ligation of protein domains. Directed evolution is among the most promising technologies used to advance this technology. However, the available screening systems for protein‐splicing act...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771659/ https://www.ncbi.nlm.nih.gov/pubmed/30963690 http://dx.doi.org/10.1002/cbic.201900139 |
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author | Beyer, Hannes M. Iwaï, Hideo |
author_facet | Beyer, Hannes M. Iwaï, Hideo |
author_sort | Beyer, Hannes M. |
collection | PubMed |
description | Protein‐splicing domains are frequently used engineering tools that find application in the in vivo and in vitro ligation of protein domains. Directed evolution is among the most promising technologies used to advance this technology. However, the available screening systems for protein‐splicing activity are associated with bottlenecks such as the selection of pseudo‐positive clones arising from off‐pathway reaction products or fragment complementation. Herein, we report a stringent screening method for protein‐splicing activity in cis and trans, that exclusively selects productively splicing domains. By fusing splicing domains to an intrinsically disordered region of the antidote from the Escherichia coli CcdA/CcdB type II toxin/antitoxin system, we linked protein splicing to cell survival. The screen allows selecting novel cis‐ and trans‐splicing inteins catalyzing productive highly efficient protein splicing, for example, from directed‐evolution approaches or the natural intein sequence space. |
format | Online Article Text |
id | pubmed-6771659 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-67716592019-10-07 Off‐Pathway‐Sensitive Protein‐Splicing Screening Based on a Toxin/Antitoxin System Beyer, Hannes M. Iwaï, Hideo Chembiochem Communications Protein‐splicing domains are frequently used engineering tools that find application in the in vivo and in vitro ligation of protein domains. Directed evolution is among the most promising technologies used to advance this technology. However, the available screening systems for protein‐splicing activity are associated with bottlenecks such as the selection of pseudo‐positive clones arising from off‐pathway reaction products or fragment complementation. Herein, we report a stringent screening method for protein‐splicing activity in cis and trans, that exclusively selects productively splicing domains. By fusing splicing domains to an intrinsically disordered region of the antidote from the Escherichia coli CcdA/CcdB type II toxin/antitoxin system, we linked protein splicing to cell survival. The screen allows selecting novel cis‐ and trans‐splicing inteins catalyzing productive highly efficient protein splicing, for example, from directed‐evolution approaches or the natural intein sequence space. John Wiley and Sons Inc. 2019-07-03 2019-08-01 /pmc/articles/PMC6771659/ /pubmed/30963690 http://dx.doi.org/10.1002/cbic.201900139 Text en © 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. |
spellingShingle | Communications Beyer, Hannes M. Iwaï, Hideo Off‐Pathway‐Sensitive Protein‐Splicing Screening Based on a Toxin/Antitoxin System |
title | Off‐Pathway‐Sensitive Protein‐Splicing Screening Based on a Toxin/Antitoxin System |
title_full | Off‐Pathway‐Sensitive Protein‐Splicing Screening Based on a Toxin/Antitoxin System |
title_fullStr | Off‐Pathway‐Sensitive Protein‐Splicing Screening Based on a Toxin/Antitoxin System |
title_full_unstemmed | Off‐Pathway‐Sensitive Protein‐Splicing Screening Based on a Toxin/Antitoxin System |
title_short | Off‐Pathway‐Sensitive Protein‐Splicing Screening Based on a Toxin/Antitoxin System |
title_sort | off‐pathway‐sensitive protein‐splicing screening based on a toxin/antitoxin system |
topic | Communications |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771659/ https://www.ncbi.nlm.nih.gov/pubmed/30963690 http://dx.doi.org/10.1002/cbic.201900139 |
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