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Construction and analysis of a genetically tuneable lytic phage display system

The Bacteriophage λ capsid protein gpD has been used extensively for fusion polypeptides that can be expressed from plasmids in Escherichia coli and remain soluble. In this study, a genetically controlled dual expression system for the display of enhanced green fluorescent protein (eGFP) was develop...

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Autores principales: Nicastro, Jessica, Sheldon, Katlyn, El-zarkout, Farah A., Sokolenko, Stanislav, Aucoin, Marc G., Slavcev, Roderick
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Berlin Heidelberg 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3745828/
https://www.ncbi.nlm.nih.gov/pubmed/23640362
http://dx.doi.org/10.1007/s00253-013-4898-6
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author Nicastro, Jessica
Sheldon, Katlyn
El-zarkout, Farah A.
Sokolenko, Stanislav
Aucoin, Marc G.
Slavcev, Roderick
author_facet Nicastro, Jessica
Sheldon, Katlyn
El-zarkout, Farah A.
Sokolenko, Stanislav
Aucoin, Marc G.
Slavcev, Roderick
author_sort Nicastro, Jessica
collection PubMed
description The Bacteriophage λ capsid protein gpD has been used extensively for fusion polypeptides that can be expressed from plasmids in Escherichia coli and remain soluble. In this study, a genetically controlled dual expression system for the display of enhanced green fluorescent protein (eGFP) was developed and characterized. Wild-type D protein (gpD) expression is encoded by λ Dam15 infecting phage particles, which can only produce a functional gpD protein when translated in amber suppressor strains of E. coli in the absence of complementing gpD from a plasmid. However, the isogenic suppressors vary dramatically in their ability to restore functional packaging to λDam15, imparting the first dimension of decorative control. In combination, the D-fusion protein, gpD::eGFP, was supplied in trans from a multicopy temperature-inducible expression plasmid, influencing D::eGFP expression and hence the availability of gpD::eGFP to complement for the Dam15 mutation and decorate viable phage progeny. Despite being the worst suppressor, maximal incorporation of gpD::eGFP into the λDam15 phage capsid was imparted by the SupD strain, conferring a gpDQ68S substitution, induced for plasmid expression of pD::eGFP. Differences in size, fluorescence and absolute protein decoration between phage preparations could be achieved by varying the temperature of and the suppressor host carrying the pD::eGFP plasmid. The effective preparation with these two variables provides a simple means by which to manage fusion decoration on the surface of phage λ.
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spelling pubmed-37458282013-08-20 Construction and analysis of a genetically tuneable lytic phage display system Nicastro, Jessica Sheldon, Katlyn El-zarkout, Farah A. Sokolenko, Stanislav Aucoin, Marc G. Slavcev, Roderick Appl Microbiol Biotechnol Applied Genetics and Molecular Biotechnology The Bacteriophage λ capsid protein gpD has been used extensively for fusion polypeptides that can be expressed from plasmids in Escherichia coli and remain soluble. In this study, a genetically controlled dual expression system for the display of enhanced green fluorescent protein (eGFP) was developed and characterized. Wild-type D protein (gpD) expression is encoded by λ Dam15 infecting phage particles, which can only produce a functional gpD protein when translated in amber suppressor strains of E. coli in the absence of complementing gpD from a plasmid. However, the isogenic suppressors vary dramatically in their ability to restore functional packaging to λDam15, imparting the first dimension of decorative control. In combination, the D-fusion protein, gpD::eGFP, was supplied in trans from a multicopy temperature-inducible expression plasmid, influencing D::eGFP expression and hence the availability of gpD::eGFP to complement for the Dam15 mutation and decorate viable phage progeny. Despite being the worst suppressor, maximal incorporation of gpD::eGFP into the λDam15 phage capsid was imparted by the SupD strain, conferring a gpDQ68S substitution, induced for plasmid expression of pD::eGFP. Differences in size, fluorescence and absolute protein decoration between phage preparations could be achieved by varying the temperature of and the suppressor host carrying the pD::eGFP plasmid. The effective preparation with these two variables provides a simple means by which to manage fusion decoration on the surface of phage λ. Springer Berlin Heidelberg 2013-05-03 2013 /pmc/articles/PMC3745828/ /pubmed/23640362 http://dx.doi.org/10.1007/s00253-013-4898-6 Text en © The Author(s) 2013 https://creativecommons.org/licenses/by-nc/2.0/ Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
spellingShingle Applied Genetics and Molecular Biotechnology
Nicastro, Jessica
Sheldon, Katlyn
El-zarkout, Farah A.
Sokolenko, Stanislav
Aucoin, Marc G.
Slavcev, Roderick
Construction and analysis of a genetically tuneable lytic phage display system
title Construction and analysis of a genetically tuneable lytic phage display system
title_full Construction and analysis of a genetically tuneable lytic phage display system
title_fullStr Construction and analysis of a genetically tuneable lytic phage display system
title_full_unstemmed Construction and analysis of a genetically tuneable lytic phage display system
title_short Construction and analysis of a genetically tuneable lytic phage display system
title_sort construction and analysis of a genetically tuneable lytic phage display system
topic Applied Genetics and Molecular Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3745828/
https://www.ncbi.nlm.nih.gov/pubmed/23640362
http://dx.doi.org/10.1007/s00253-013-4898-6
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