Determination of In Vivo Interactomes of Dot/Icm Type IV Secretion System Effectors by Tandem Affinity Purification

Ernest C So, Aurélie Mousnier, Gad Frankel, Gunnar N Schroeder

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)

Abstract

The Dot/Icm type IV secretion system (T4SS) is essential for the pathogenesis of Legionella species and translocates a multitude of effector proteins into host cells. The identification of host cell targets of these effectors is often critical to unravel their roles in controlling the host. Here we describe a method to characterize the protein complexes associated with effectors in infected host cells. To achieve this, Legionella expressing an effector of interest fused to a Bio-tag, a combination of hexahistidine tags and a specific recognition sequence for the biotin ligase BirA, are used to infect host cells expressing BirA, which leads to biotinylation of the translocated effector. Following chemical cross-linking, effector interactomes are isolated by tandem affinity purification employing metal affinity and NeutrAvidin resins and identified by western blotting or mass spectrometry.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
Subtitle of host publicationLegionella
PublisherSpringer
Pages289-303
Number of pages15
Volume1921
DOIs
Publication statusPublished - 30 Jan 2019

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
ISSN (Print)1064-3745

Fingerprint

Legionella
His-His-His-His-His-His
Biotinylation
Ligases
Biotin
Mass Spectrometry
Proteins
Western Blotting
Metals
Type IV Secretion Systems
neutravidin

Cite this

So, E. C., Mousnier, A., Frankel, G., & Schroeder, G. N. (2019). Determination of In Vivo Interactomes of Dot/Icm Type IV Secretion System Effectors by Tandem Affinity Purification. In Methods in Molecular Biology : Legionella (Vol. 1921, pp. 289-303). (Methods in Molecular Biology ). Springer. https://doi.org/10.1007/978-1-4939-9048-1_19
So, Ernest C ; Mousnier, Aurélie ; Frankel, Gad ; Schroeder, Gunnar N. / Determination of In Vivo Interactomes of Dot/Icm Type IV Secretion System Effectors by Tandem Affinity Purification. Methods in Molecular Biology : Legionella . Vol. 1921 Springer, 2019. pp. 289-303 (Methods in Molecular Biology ).
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abstract = "The Dot/Icm type IV secretion system (T4SS) is essential for the pathogenesis of Legionella species and translocates a multitude of effector proteins into host cells. The identification of host cell targets of these effectors is often critical to unravel their roles in controlling the host. Here we describe a method to characterize the protein complexes associated with effectors in infected host cells. To achieve this, Legionella expressing an effector of interest fused to a Bio-tag, a combination of hexahistidine tags and a specific recognition sequence for the biotin ligase BirA, are used to infect host cells expressing BirA, which leads to biotinylation of the translocated effector. Following chemical cross-linking, effector interactomes are isolated by tandem affinity purification employing metal affinity and NeutrAvidin resins and identified by western blotting or mass spectrometry.",
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So, EC, Mousnier, A, Frankel, G & Schroeder, GN 2019, Determination of In Vivo Interactomes of Dot/Icm Type IV Secretion System Effectors by Tandem Affinity Purification. in Methods in Molecular Biology : Legionella . vol. 1921, Methods in Molecular Biology , Springer, pp. 289-303. https://doi.org/10.1007/978-1-4939-9048-1_19

Determination of In Vivo Interactomes of Dot/Icm Type IV Secretion System Effectors by Tandem Affinity Purification. / So, Ernest C; Mousnier, Aurélie; Frankel, Gad; Schroeder, Gunnar N.

Methods in Molecular Biology : Legionella . Vol. 1921 Springer, 2019. p. 289-303 (Methods in Molecular Biology ).

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)

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AB - The Dot/Icm type IV secretion system (T4SS) is essential for the pathogenesis of Legionella species and translocates a multitude of effector proteins into host cells. The identification of host cell targets of these effectors is often critical to unravel their roles in controlling the host. Here we describe a method to characterize the protein complexes associated with effectors in infected host cells. To achieve this, Legionella expressing an effector of interest fused to a Bio-tag, a combination of hexahistidine tags and a specific recognition sequence for the biotin ligase BirA, are used to infect host cells expressing BirA, which leads to biotinylation of the translocated effector. Following chemical cross-linking, effector interactomes are isolated by tandem affinity purification employing metal affinity and NeutrAvidin resins and identified by western blotting or mass spectrometry.

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M3 - Chapter (peer-reviewed)

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VL - 1921

T3 - Methods in Molecular Biology

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So EC, Mousnier A, Frankel G, Schroeder GN. Determination of In Vivo Interactomes of Dot/Icm Type IV Secretion System Effectors by Tandem Affinity Purification. In Methods in Molecular Biology : Legionella . Vol. 1921. Springer. 2019. p. 289-303. (Methods in Molecular Biology ). https://doi.org/10.1007/978-1-4939-9048-1_19