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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...

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Detalles Bibliográficos
Autores principales: Beyer, Hannes M., Iwaï, Hideo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2019
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.
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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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