Cargando…

Sequence-based prediction of permissive stretches for internal protein tagging and knockdown

BACKGROUND: Internal tagging of proteins by inserting small functional peptides into surface accessible permissive sites has proven to be an indispensable tool for basic and applied science. Permissive sites are typically identified by transposon mutagenesis on a case-by-case basis, limiting scalabi...

Descripción completa

Detalles Bibliográficos
Autores principales: Oesterle, Sabine, Roberts, Tania Michelle, Widmer, Lukas Andreas, Mustafa, Harun, Panke, Sven, Billerbeck, Sonja
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5661948/
https://www.ncbi.nlm.nih.gov/pubmed/29084520
http://dx.doi.org/10.1186/s12915-017-0440-0
_version_ 1783274569583296512
author Oesterle, Sabine
Roberts, Tania Michelle
Widmer, Lukas Andreas
Mustafa, Harun
Panke, Sven
Billerbeck, Sonja
author_facet Oesterle, Sabine
Roberts, Tania Michelle
Widmer, Lukas Andreas
Mustafa, Harun
Panke, Sven
Billerbeck, Sonja
author_sort Oesterle, Sabine
collection PubMed
description BACKGROUND: Internal tagging of proteins by inserting small functional peptides into surface accessible permissive sites has proven to be an indispensable tool for basic and applied science. Permissive sites are typically identified by transposon mutagenesis on a case-by-case basis, limiting scalability and their exploitation as a system-wide protein engineering tool. METHODS: We developed an apporach for predicting permissive stretches (PSs) in proteins based on the identification of length-variable regions (regions containing indels) in homologous proteins. RESULTS: We verify that a protein's primary structure information alone is sufficient to identify PSs. Identified PSs are predicted to be predominantly surface accessible; hence, the position of inserted peptides is likely suitable for diverse applications. We demonstrate the viability of this approach by inserting a Tobacco etch virus protease recognition site (TEV-tag) into several PSs in a wide range of proteins, from small monomeric enzymes (adenylate kinase) to large multi-subunit molecular machines (ATP synthase) and verify their functionality after insertion. We apply this method to engineer conditional protein knockdowns directly in the Escherichia coli chromosome and generate a cell-free platform with enhanced nucleotide stability. CONCLUSIONS: Functional internally tagged proteins can be rationally designed and directly chromosomally implemented. Critical for the successful design of protein knockdowns was the incorporation of surface accessibility and secondary structure predictions, as well as the design of an improved TEV-tag that enables efficient hydrolysis when inserted into the middle of a protein. This versatile and portable approach can likely be adapted for other applications, and broadly adopted. We provide guidelines for the design of internally tagged proteins in order to empower scientists with little or no protein engineering expertise to internally tag their target proteins. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12915-017-0440-0) contains supplementary material, which is available to authorized users.
format Online
Article
Text
id pubmed-5661948
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-56619482017-11-01 Sequence-based prediction of permissive stretches for internal protein tagging and knockdown Oesterle, Sabine Roberts, Tania Michelle Widmer, Lukas Andreas Mustafa, Harun Panke, Sven Billerbeck, Sonja BMC Biol Methodology Article BACKGROUND: Internal tagging of proteins by inserting small functional peptides into surface accessible permissive sites has proven to be an indispensable tool for basic and applied science. Permissive sites are typically identified by transposon mutagenesis on a case-by-case basis, limiting scalability and their exploitation as a system-wide protein engineering tool. METHODS: We developed an apporach for predicting permissive stretches (PSs) in proteins based on the identification of length-variable regions (regions containing indels) in homologous proteins. RESULTS: We verify that a protein's primary structure information alone is sufficient to identify PSs. Identified PSs are predicted to be predominantly surface accessible; hence, the position of inserted peptides is likely suitable for diverse applications. We demonstrate the viability of this approach by inserting a Tobacco etch virus protease recognition site (TEV-tag) into several PSs in a wide range of proteins, from small monomeric enzymes (adenylate kinase) to large multi-subunit molecular machines (ATP synthase) and verify their functionality after insertion. We apply this method to engineer conditional protein knockdowns directly in the Escherichia coli chromosome and generate a cell-free platform with enhanced nucleotide stability. CONCLUSIONS: Functional internally tagged proteins can be rationally designed and directly chromosomally implemented. Critical for the successful design of protein knockdowns was the incorporation of surface accessibility and secondary structure predictions, as well as the design of an improved TEV-tag that enables efficient hydrolysis when inserted into the middle of a protein. This versatile and portable approach can likely be adapted for other applications, and broadly adopted. We provide guidelines for the design of internally tagged proteins in order to empower scientists with little or no protein engineering expertise to internally tag their target proteins. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12915-017-0440-0) contains supplementary material, which is available to authorized users. BioMed Central 2017-10-30 /pmc/articles/PMC5661948/ /pubmed/29084520 http://dx.doi.org/10.1186/s12915-017-0440-0 Text en © Panke et al. 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Methodology Article
Oesterle, Sabine
Roberts, Tania Michelle
Widmer, Lukas Andreas
Mustafa, Harun
Panke, Sven
Billerbeck, Sonja
Sequence-based prediction of permissive stretches for internal protein tagging and knockdown
title Sequence-based prediction of permissive stretches for internal protein tagging and knockdown
title_full Sequence-based prediction of permissive stretches for internal protein tagging and knockdown
title_fullStr Sequence-based prediction of permissive stretches for internal protein tagging and knockdown
title_full_unstemmed Sequence-based prediction of permissive stretches for internal protein tagging and knockdown
title_short Sequence-based prediction of permissive stretches for internal protein tagging and knockdown
title_sort sequence-based prediction of permissive stretches for internal protein tagging and knockdown
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5661948/
https://www.ncbi.nlm.nih.gov/pubmed/29084520
http://dx.doi.org/10.1186/s12915-017-0440-0
work_keys_str_mv AT oesterlesabine sequencebasedpredictionofpermissivestretchesforinternalproteintaggingandknockdown
AT robertstaniamichelle sequencebasedpredictionofpermissivestretchesforinternalproteintaggingandknockdown
AT widmerlukasandreas sequencebasedpredictionofpermissivestretchesforinternalproteintaggingandknockdown
AT mustafaharun sequencebasedpredictionofpermissivestretchesforinternalproteintaggingandknockdown
AT pankesven sequencebasedpredictionofpermissivestretchesforinternalproteintaggingandknockdown
AT billerbecksonja sequencebasedpredictionofpermissivestretchesforinternalproteintaggingandknockdown