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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 MAVS-TRAF3 interactions and regulation by HCV NS3/4A in HEK293 cells.(A) HAVI-Fluc-MAVS was determined to be associated with Rluc-TRAF3 using the DLR-PD assay. HEK293 cells were co-transfected with the indicated vectors. The percentage of Rluc on the beads compared with the cell lysate was calculated by dividing the Rluc activity of Rluc-TRAF3 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. (B) Using the DLR-PD assay, the HAVI-Fluc-MAVS association with Rluc-TRAF3 was determined to be inhibited by HCV NS3/4A in a dose-dependent manner in HEK293 cells. Cells were co-transfected with HAVI-Fluc-MAVS (100 ng), Rluc-TRAF3 (100 ng) and DNA (100 ng) containing the NS3/4A vector (0, 10, 50 or 100 ng) and control plasmid pCI neo. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads. (C) Biotinylated protein pull-down assay to determine the MAVS-TRAF3 interactions and regulation by HCV NS3/4A in HEK293 cells. (D) Overexpression of HCV NS3/4A inhibited the MAVS-induced activation of the IFNβ-Luc promoter after normalization with RL-TK in HEK293 cells. The Fluc activity from different cell lysates was divided by that of cell lysates from mock transfected cells without MAVS activation.
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pone-0026414-g007: Analysis of MAVS-TRAF3 interactions and regulation by HCV NS3/4A in HEK293 cells.(A) HAVI-Fluc-MAVS was determined to be associated with Rluc-TRAF3 using the DLR-PD assay. HEK293 cells were co-transfected with the indicated vectors. The percentage of Rluc on the beads compared with the cell lysate was calculated by dividing the Rluc activity of Rluc-TRAF3 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. (B) Using the DLR-PD assay, the HAVI-Fluc-MAVS association with Rluc-TRAF3 was determined to be inhibited by HCV NS3/4A in a dose-dependent manner in HEK293 cells. Cells were co-transfected with HAVI-Fluc-MAVS (100 ng), Rluc-TRAF3 (100 ng) and DNA (100 ng) containing the NS3/4A vector (0, 10, 50 or 100 ng) and control plasmid pCI neo. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads. (C) Biotinylated protein pull-down assay to determine the MAVS-TRAF3 interactions and regulation by HCV NS3/4A in HEK293 cells. (D) Overexpression of HCV NS3/4A inhibited the MAVS-induced activation of the IFNβ-Luc promoter after normalization with RL-TK in HEK293 cells. The Fluc activity from different cell lysates was divided by that of cell lysates from mock transfected cells without MAVS activation.

Mentions: TRAF3 has been shown to be a downstream molecule of MAVS in the RIG-I-MAVS cytoplasmic antiviral signal pathway. Specific interactions between MAVS-TRAF3 regulate the signaling of MAVS [19]. To quantitatively measure MAVS-TRAF3 interactions, HAVI-Fluc-MAVS was co-overexpressed with Rluc-TRAF3 in HEK293 cells, and the DLR-PD assay was performed. The percentage of Rluc on the beads compared with the cell lysate was 1.85% (Fig. 7A), which is more than 26-fold higher than that of three negative controls (P<0.05) and higher than the cutoff value of 3-fold for positive PPIs [6]. These results suggest that MAVS interacts with TRAF3. Additionally, interactions between MAVS and TRAF3 were also confirmed by a traditional pull-down assay (Fig. 7C). The results of the DLR-PD assay demonstrated that the activity ratio of Rluc/Fluc to Rluc-TRAF3 and HAVI-Fluc-MAVS on the beads was approximately 2.29∶1(Fig. 7A), indicating the relative amount of interacting proteins.


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 MAVS-TRAF3 interactions and regulation by HCV NS3/4A in HEK293 cells.(A) HAVI-Fluc-MAVS was determined to be associated with Rluc-TRAF3 using the DLR-PD assay. HEK293 cells were co-transfected with the indicated vectors. The percentage of Rluc on the beads compared with the cell lysate was calculated by dividing the Rluc activity of Rluc-TRAF3 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. (B) Using the DLR-PD assay, the HAVI-Fluc-MAVS association with Rluc-TRAF3 was determined to be inhibited by HCV NS3/4A in a dose-dependent manner in HEK293 cells. Cells were co-transfected with HAVI-Fluc-MAVS (100 ng), Rluc-TRAF3 (100 ng) and DNA (100 ng) containing the NS3/4A vector (0, 10, 50 or 100 ng) and control plasmid pCI neo. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads. (C) Biotinylated protein pull-down assay to determine the MAVS-TRAF3 interactions and regulation by HCV NS3/4A in HEK293 cells. (D) Overexpression of HCV NS3/4A inhibited the MAVS-induced activation of the IFNβ-Luc promoter after normalization with RL-TK in HEK293 cells. The Fluc activity from different cell lysates was divided by that of cell lysates from mock transfected cells without MAVS activation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198426&req=5

pone-0026414-g007: Analysis of MAVS-TRAF3 interactions and regulation by HCV NS3/4A in HEK293 cells.(A) HAVI-Fluc-MAVS was determined to be associated with Rluc-TRAF3 using the DLR-PD assay. HEK293 cells were co-transfected with the indicated vectors. The percentage of Rluc on the beads compared with the cell lysate was calculated by dividing the Rluc activity of Rluc-TRAF3 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. (B) Using the DLR-PD assay, the HAVI-Fluc-MAVS association with Rluc-TRAF3 was determined to be inhibited by HCV NS3/4A in a dose-dependent manner in HEK293 cells. Cells were co-transfected with HAVI-Fluc-MAVS (100 ng), Rluc-TRAF3 (100 ng) and DNA (100 ng) containing the NS3/4A vector (0, 10, 50 or 100 ng) and control plasmid pCI neo. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads. (C) Biotinylated protein pull-down assay to determine the MAVS-TRAF3 interactions and regulation by HCV NS3/4A in HEK293 cells. (D) Overexpression of HCV NS3/4A inhibited the MAVS-induced activation of the IFNβ-Luc promoter after normalization with RL-TK in HEK293 cells. The Fluc activity from different cell lysates was divided by that of cell lysates from mock transfected cells without MAVS activation.
Mentions: TRAF3 has been shown to be a downstream molecule of MAVS in the RIG-I-MAVS cytoplasmic antiviral signal pathway. Specific interactions between MAVS-TRAF3 regulate the signaling of MAVS [19]. To quantitatively measure MAVS-TRAF3 interactions, HAVI-Fluc-MAVS was co-overexpressed with Rluc-TRAF3 in HEK293 cells, and the DLR-PD assay was performed. The percentage of Rluc on the beads compared with the cell lysate was 1.85% (Fig. 7A), which is more than 26-fold higher than that of three negative controls (P<0.05) and higher than the cutoff value of 3-fold for positive PPIs [6]. These results suggest that MAVS interacts with TRAF3. Additionally, interactions between MAVS and TRAF3 were also confirmed by a traditional pull-down assay (Fig. 7C). The results of the DLR-PD assay demonstrated that the activity ratio of Rluc/Fluc to Rluc-TRAF3 and HAVI-Fluc-MAVS on the beads was approximately 2.29∶1(Fig. 7A), indicating the relative amount of interacting proteins.

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