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Relative quantification of protein-protein interactions using a dual luciferase reporter pull-down assay system.

Jia S, Peng J, Gao B, Chen Z, Zhou Y, Fu Q, Wang H, Zhan L - PLoS ONE (2011)

Bottom Line: The lack of simple, sensitive approaches to precisely quantify protein-protein interactions still prevents our understanding of the functions of many proteins.This study provides a simple, rapid, sensitive, and efficient approach to identify and quantify relative protein-protein interactions.Importantly, the dual luciferase reporter pull-down method will facilitate the functional determination of proteins.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Transfusion Medicine, Beijing, China.

ABSTRACT
The identification and quantitative analysis of protein-protein interactions are essential to the functional characterization of proteins in the post-proteomics era. The methods currently available are generally time-consuming, technically complicated, insensitive and/or semi-quantitative. The lack of simple, sensitive approaches to precisely quantify protein-protein interactions still prevents our understanding of the functions of many proteins. Here, we develop a novel dual luciferase reporter pull-down assay by combining a biotinylated Firefly luciferase pull-down assay with a dual luciferase reporter assay. The biotinylated Firefly luciferase-tagged protein enables rapid and efficient isolation of a putative Renilla luciferase-tagged binding protein from a relatively small amount of sample. Both of these proteins can be quantitatively detected using the dual luciferase reporter assay system. Protein-protein interactions, including Fos-Jun located in the nucleus; MAVS-TRAF3 in cytoplasm; inducible IRF3 dimerization; viral protein-regulated interactions, such as MAVS-MAVS and MAVS-TRAF3; IRF3 dimerization; and protein interaction domain mapping, are studied using this novel assay system. Herein, we demonstrate that this dual luciferase reporter pull-down assay enables the quantification of the relative amounts of interacting proteins that bind to streptavidin-coupled beads for protein purification. This study provides a simple, rapid, sensitive, and efficient approach to identify and quantify relative protein-protein interactions. Importantly, the dual luciferase reporter pull-down method will facilitate the functional determination of proteins.

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Analysis of IRF3 dimerization in the innate antiviral signaling pathway in HEK293 cells.(A) HAVI-Fluc-IRF3 did not associate with Rluc-IRF3 using the DLR-PD assay. HEK293 cells were co-transfected with the indicated expression vectors (100 ng), and the DLR-PD assay was performed. The Fluc and Rluc activity in lysate and on the beads were measured. The ratio of Fluc and Rluc activity on the beads compared with the cell lysate was calculated by dividing the Fluc and Rluc activity measured on the beads by the Fluc and Rluc activity of the same amount of lysate used in the pull-down assay. The percentage of Rluc on the beads compared with the cell lysate was calculated by dividing the Rluc activity measured on the beads by the Rluc activity of the same amount of lysate used in the pull-down assay. (B) HAVI-Fluc-IRF3 association with Rluc-IRF3 induced by overexpressed eYFP-MAVS in a dose-dependent manner using the DLR-PD assay. HEK293 cells were co-transfected with HAVI-Fluc-IRF3 (100 ng), Rluc-IRF3 (100 ng), and DNA (100 ng) containing eYFP-MAVS vector (0, 10, 50 or 100 ng) and control plasmid pcDNA3.1. The percentage of Rluc on the beads compared with the lysate was calculated by dividing the Rluc activity of Rluc-IRF3 measured on the beads by the Rluc activity in the same amount of lysate used in the pull-down assay. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads. (C) HAVI-Fluc-IRF3 association with Rluc-IRF3 induced by overexpressed eYFP-MAVS in HEK293 cells using the biotinylated protein pull-down assay. (D) Evaluation of the HAVI-IRF3 association with Myc-IRF3 induced by overexpressed eYFP-MAVS in HEK293 cells using the biotinylated protein pull-down assay. (E) The curve of the Rluc/Fluc ratio on the beads based on the DLR-PD assay results. HEK293 cells were transfected with eYFP-MAVS (50 ng), HAVI-Fluc-IRF3 (20 ng) or HAVI-Fluc (20 ng) and Rluc-IRF3 plasmid DNA (1, 10, 100 or 500 ng). Cells were collected at 48 h post-transfection and subsequently used in the DLR-PD assay.
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pone-0026414-g004: Analysis of IRF3 dimerization in the innate antiviral signaling pathway in HEK293 cells.(A) HAVI-Fluc-IRF3 did not associate with Rluc-IRF3 using the DLR-PD assay. HEK293 cells were co-transfected with the indicated expression vectors (100 ng), and the DLR-PD assay was performed. The Fluc and Rluc activity in lysate and on the beads were measured. The ratio of Fluc and Rluc activity on the beads compared with the cell lysate was calculated by dividing the Fluc and Rluc activity measured on the beads by the Fluc and Rluc activity of the same amount of lysate used in the pull-down assay. The percentage of Rluc on the beads compared with the cell lysate was calculated by dividing the Rluc activity measured on the beads by the Rluc activity of the same amount of lysate used in the pull-down assay. (B) HAVI-Fluc-IRF3 association with Rluc-IRF3 induced by overexpressed eYFP-MAVS in a dose-dependent manner using the DLR-PD assay. HEK293 cells were co-transfected with HAVI-Fluc-IRF3 (100 ng), Rluc-IRF3 (100 ng), and DNA (100 ng) containing eYFP-MAVS vector (0, 10, 50 or 100 ng) and control plasmid pcDNA3.1. The percentage of Rluc on the beads compared with the lysate was calculated by dividing the Rluc activity of Rluc-IRF3 measured on the beads by the Rluc activity in the same amount of lysate used in the pull-down assay. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads. (C) HAVI-Fluc-IRF3 association with Rluc-IRF3 induced by overexpressed eYFP-MAVS in HEK293 cells using the biotinylated protein pull-down assay. (D) Evaluation of the HAVI-IRF3 association with Myc-IRF3 induced by overexpressed eYFP-MAVS in HEK293 cells using the biotinylated protein pull-down assay. (E) The curve of the Rluc/Fluc ratio on the beads based on the DLR-PD assay results. HEK293 cells were transfected with eYFP-MAVS (50 ng), HAVI-Fluc-IRF3 (20 ng) or HAVI-Fluc (20 ng) and Rluc-IRF3 plasmid DNA (1, 10, 100 or 500 ng). Cells were collected at 48 h post-transfection and subsequently used in the DLR-PD assay.

Mentions: To relatively quantify IRF3 dimerization via the DLR-PD assay, IRF3 was fused with a biotinylated Fluc tag and a Rluc tag. Even though HAVI-Fluc-IRF3 was co-overexpressed with Rluc-IRF3 in HEK293 cells, the percentage of Rluc on beads compared with the cell lysate was close to that of three negative controls (P>0.05)(Fig. 4A). These data suggest that IRF3 does not dimerize without stimulation.


Relative quantification of protein-protein interactions using a dual luciferase reporter pull-down assay system.

Jia S, Peng J, Gao B, Chen Z, Zhou Y, Fu Q, Wang H, Zhan L - PLoS ONE (2011)

Analysis of IRF3 dimerization in the innate antiviral signaling pathway in HEK293 cells.(A) HAVI-Fluc-IRF3 did not associate with Rluc-IRF3 using the DLR-PD assay. HEK293 cells were co-transfected with the indicated expression vectors (100 ng), and the DLR-PD assay was performed. The Fluc and Rluc activity in lysate and on the beads were measured. The ratio of Fluc and Rluc activity on the beads compared with the cell lysate was calculated by dividing the Fluc and Rluc activity measured on the beads by the Fluc and Rluc activity of the same amount of lysate used in the pull-down assay. The percentage of Rluc on the beads compared with the cell lysate was calculated by dividing the Rluc activity measured on the beads by the Rluc activity of the same amount of lysate used in the pull-down assay. (B) HAVI-Fluc-IRF3 association with Rluc-IRF3 induced by overexpressed eYFP-MAVS in a dose-dependent manner using the DLR-PD assay. HEK293 cells were co-transfected with HAVI-Fluc-IRF3 (100 ng), Rluc-IRF3 (100 ng), and DNA (100 ng) containing eYFP-MAVS vector (0, 10, 50 or 100 ng) and control plasmid pcDNA3.1. The percentage of Rluc on the beads compared with the lysate was calculated by dividing the Rluc activity of Rluc-IRF3 measured on the beads by the Rluc activity in the same amount of lysate used in the pull-down assay. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads. (C) HAVI-Fluc-IRF3 association with Rluc-IRF3 induced by overexpressed eYFP-MAVS in HEK293 cells using the biotinylated protein pull-down assay. (D) Evaluation of the HAVI-IRF3 association with Myc-IRF3 induced by overexpressed eYFP-MAVS in HEK293 cells using the biotinylated protein pull-down assay. (E) The curve of the Rluc/Fluc ratio on the beads based on the DLR-PD assay results. HEK293 cells were transfected with eYFP-MAVS (50 ng), HAVI-Fluc-IRF3 (20 ng) or HAVI-Fluc (20 ng) and Rluc-IRF3 plasmid DNA (1, 10, 100 or 500 ng). Cells were collected at 48 h post-transfection and subsequently used in the DLR-PD assay.
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Related In: Results  -  Collection

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pone-0026414-g004: Analysis of IRF3 dimerization in the innate antiviral signaling pathway in HEK293 cells.(A) HAVI-Fluc-IRF3 did not associate with Rluc-IRF3 using the DLR-PD assay. HEK293 cells were co-transfected with the indicated expression vectors (100 ng), and the DLR-PD assay was performed. The Fluc and Rluc activity in lysate and on the beads were measured. The ratio of Fluc and Rluc activity on the beads compared with the cell lysate was calculated by dividing the Fluc and Rluc activity measured on the beads by the Fluc and Rluc activity of the same amount of lysate used in the pull-down assay. The percentage of Rluc on the beads compared with the cell lysate was calculated by dividing the Rluc activity measured on the beads by the Rluc activity of the same amount of lysate used in the pull-down assay. (B) HAVI-Fluc-IRF3 association with Rluc-IRF3 induced by overexpressed eYFP-MAVS in a dose-dependent manner using the DLR-PD assay. HEK293 cells were co-transfected with HAVI-Fluc-IRF3 (100 ng), Rluc-IRF3 (100 ng), and DNA (100 ng) containing eYFP-MAVS vector (0, 10, 50 or 100 ng) and control plasmid pcDNA3.1. The percentage of Rluc on the beads compared with the lysate was calculated by dividing the Rluc activity of Rluc-IRF3 measured on the beads by the Rluc activity in the same amount of lysate used in the pull-down assay. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads. (C) HAVI-Fluc-IRF3 association with Rluc-IRF3 induced by overexpressed eYFP-MAVS in HEK293 cells using the biotinylated protein pull-down assay. (D) Evaluation of the HAVI-IRF3 association with Myc-IRF3 induced by overexpressed eYFP-MAVS in HEK293 cells using the biotinylated protein pull-down assay. (E) The curve of the Rluc/Fluc ratio on the beads based on the DLR-PD assay results. HEK293 cells were transfected with eYFP-MAVS (50 ng), HAVI-Fluc-IRF3 (20 ng) or HAVI-Fluc (20 ng) and Rluc-IRF3 plasmid DNA (1, 10, 100 or 500 ng). Cells were collected at 48 h post-transfection and subsequently used in the DLR-PD assay.
Mentions: To relatively quantify IRF3 dimerization via the DLR-PD assay, IRF3 was fused with a biotinylated Fluc tag and a Rluc tag. Even though HAVI-Fluc-IRF3 was co-overexpressed with Rluc-IRF3 in HEK293 cells, the percentage of Rluc on beads compared with the cell lysate was close to that of three negative controls (P>0.05)(Fig. 4A). These data suggest that IRF3 does not dimerize without stimulation.

Bottom Line: The lack of simple, sensitive approaches to precisely quantify protein-protein interactions still prevents our understanding of the functions of many proteins.This study provides a simple, rapid, sensitive, and efficient approach to identify and quantify relative protein-protein interactions.Importantly, the dual luciferase reporter pull-down method will facilitate the functional determination of proteins.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Transfusion Medicine, Beijing, China.

ABSTRACT
The identification and quantitative analysis of protein-protein interactions are essential to the functional characterization of proteins in the post-proteomics era. The methods currently available are generally time-consuming, technically complicated, insensitive and/or semi-quantitative. The lack of simple, sensitive approaches to precisely quantify protein-protein interactions still prevents our understanding of the functions of many proteins. Here, we develop a novel dual luciferase reporter pull-down assay by combining a biotinylated Firefly luciferase pull-down assay with a dual luciferase reporter assay. The biotinylated Firefly luciferase-tagged protein enables rapid and efficient isolation of a putative Renilla luciferase-tagged binding protein from a relatively small amount of sample. Both of these proteins can be quantitatively detected using the dual luciferase reporter assay system. Protein-protein interactions, including Fos-Jun located in the nucleus; MAVS-TRAF3 in cytoplasm; inducible IRF3 dimerization; viral protein-regulated interactions, such as MAVS-MAVS and MAVS-TRAF3; IRF3 dimerization; and protein interaction domain mapping, are studied using this novel assay system. Herein, we demonstrate that this dual luciferase reporter pull-down assay enables the quantification of the relative amounts of interacting proteins that bind to streptavidin-coupled beads for protein purification. This study provides a simple, rapid, sensitive, and efficient approach to identify and quantify relative protein-protein interactions. Importantly, the dual luciferase reporter pull-down method will facilitate the functional determination of proteins.

Show MeSH