Expression and purification of phage T7 ejection proteins for cryo-EM analysis

Summary: Bacteriophages of the Podoviridae family densely package their genomes into precursor capsids alongside internal virion proteins called ejection proteins. In phage T7 these proteins (gp14, gp15, and gp16) are ejected into the host envelope forming a DNA-ejectosome for genome delivery. Here,...

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Autores principales:	Nicholas A. Swanson, Ravi K. Lokareddy, Fenglin Li, Chun-feng Hou, Mikhail Pavlenok, Michael Niederweis, Gino Cingolani
Formato:	article
Lenguaje:	EN
Publicado:	Elsevier 2021
Materias:	Microbiology Microscopy Protein Biochemistry Protein expression and purification Structural Biology Cryo-EM Science (General) Q1-390
Acceso en línea:	https://doaj.org/article/01c100bce6b94e788ce7a8bc10be80d3
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spelling	oai:doaj.org-article:01c100bce6b94e788ce7a8bc10be80d32021-11-18T04:51:43ZExpression and purification of phage T7 ejection proteins for cryo-EM analysis2666-166710.1016/j.xpro.2021.100960https://doaj.org/article/01c100bce6b94e788ce7a8bc10be80d32021-12-01T00:00:00Zhttp://www.sciencedirect.com/science/article/pii/S2666166721006663https://doaj.org/toc/2666-1667Summary: Bacteriophages of the Podoviridae family densely package their genomes into precursor capsids alongside internal virion proteins called ejection proteins. In phage T7 these proteins (gp14, gp15, and gp16) are ejected into the host envelope forming a DNA-ejectosome for genome delivery. Here, we describe the purification and characterization of recombinant gp14, gp15, and gp16. This protocol was used for high-resolution cryo-EM structure analysis of the T7 periplasmic tunnel and can be adapted to study ejection proteins from other phages.For complete details on the use and execution of this protocol, please refer to Swanson et al. (2021).Nicholas A. SwansonRavi K. LokareddyFenglin LiChun-feng HouMikhail PavlenokMichael NiederweisGino CingolaniElsevierarticleMicrobiologyMicroscopyProtein BiochemistryProtein expression and purificationStructural BiologyCryo-EMScience (General)Q1-390ENSTAR Protocols, Vol 2, Iss 4, Pp 100960- (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology Microscopy Protein Biochemistry Protein expression and purification Structural Biology Cryo-EM Science (General) Q1-390
spellingShingle	Microbiology Microscopy Protein Biochemistry Protein expression and purification Structural Biology Cryo-EM Science (General) Q1-390 Nicholas A. Swanson Ravi K. Lokareddy Fenglin Li Chun-feng Hou Mikhail Pavlenok Michael Niederweis Gino Cingolani Expression and purification of phage T7 ejection proteins for cryo-EM analysis
description	Summary: Bacteriophages of the Podoviridae family densely package their genomes into precursor capsids alongside internal virion proteins called ejection proteins. In phage T7 these proteins (gp14, gp15, and gp16) are ejected into the host envelope forming a DNA-ejectosome for genome delivery. Here, we describe the purification and characterization of recombinant gp14, gp15, and gp16. This protocol was used for high-resolution cryo-EM structure analysis of the T7 periplasmic tunnel and can be adapted to study ejection proteins from other phages.For complete details on the use and execution of this protocol, please refer to Swanson et al. (2021).
format	article
author	Nicholas A. Swanson Ravi K. Lokareddy Fenglin Li Chun-feng Hou Mikhail Pavlenok Michael Niederweis Gino Cingolani
author_facet	Nicholas A. Swanson Ravi K. Lokareddy Fenglin Li Chun-feng Hou Mikhail Pavlenok Michael Niederweis Gino Cingolani
author_sort	Nicholas A. Swanson
title	Expression and purification of phage T7 ejection proteins for cryo-EM analysis
title_short	Expression and purification of phage T7 ejection proteins for cryo-EM analysis
title_full	Expression and purification of phage T7 ejection proteins for cryo-EM analysis
title_fullStr	Expression and purification of phage T7 ejection proteins for cryo-EM analysis
title_full_unstemmed	Expression and purification of phage T7 ejection proteins for cryo-EM analysis
title_sort	expression and purification of phage t7 ejection proteins for cryo-em analysis
publisher	Elsevier
publishDate	2021
url	https://doaj.org/article/01c100bce6b94e788ce7a8bc10be80d3
work_keys_str_mv	AT nicholasaswanson expressionandpurificationofphaget7ejectionproteinsforcryoemanalysis AT raviklokareddy expressionandpurificationofphaget7ejectionproteinsforcryoemanalysis AT fenglinli expressionandpurificationofphaget7ejectionproteinsforcryoemanalysis AT chunfenghou expressionandpurificationofphaget7ejectionproteinsforcryoemanalysis AT mikhailpavlenok expressionandpurificationofphaget7ejectionproteinsforcryoemanalysis AT michaelniederweis expressionandpurificationofphaget7ejectionproteinsforcryoemanalysis AT ginocingolani expressionandpurificationofphaget7ejectionproteinsforcryoemanalysis
_version_	1718424953595887616

Expression and purification of phage T7 ejection proteins for cryo-EM analysis

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