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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.

Show MeSH
Analysis of MAVS dimerization and inhibition by HCV NS3/4A in HEK293 cells.(A) Evaluation of the HAVI-Fluc-MAVS association with Rluc-MAVS using the DLR-PD assay. HEK293 cells were co-transfected with the indicated vectors (100 ng). The percentage of Rluc on the beads compared with the cell lysate was calculated by dividing the Rluc activity of Rluc-MAVS on the beads with the Rluc activity in the same amount of lysate used in the pull-down assays. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads. (B) HAVI-MAVS was cleaved by HCV NS3/4A and detected by Western blot when coexpressed in HEK293 cells. (C) Analysis of the interactions between HAVI-Fluc-MAVS and Rluc-MAVS inhibited by HCV NS3/4A using the DLR-PD assay. HEK293 cells were co-transfected with HAVI-Fluc-MAVS (100 ng), Rluc-MAVS (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.
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pone-0026414-g006: Analysis of MAVS dimerization and inhibition by HCV NS3/4A in HEK293 cells.(A) Evaluation of the HAVI-Fluc-MAVS association with Rluc-MAVS using the DLR-PD assay. HEK293 cells were co-transfected with the indicated vectors (100 ng). The percentage of Rluc on the beads compared with the cell lysate was calculated by dividing the Rluc activity of Rluc-MAVS on the beads with the Rluc activity in the same amount of lysate used in the pull-down assays. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads. (B) HAVI-MAVS was cleaved by HCV NS3/4A and detected by Western blot when coexpressed in HEK293 cells. (C) Analysis of the interactions between HAVI-Fluc-MAVS and Rluc-MAVS inhibited by HCV NS3/4A using the DLR-PD assay. HEK293 cells were co-transfected with HAVI-Fluc-MAVS (100 ng), Rluc-MAVS (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.

Mentions: To determine whether the DLR-PD assay is applicable to the analysis of membranous protein interactions, MAVS dimerization was measured using the DLR-PD assay. MAVS is located on the outer membrane of mitochondria, and its self-association mediates antiviral innate immune signaling [16], [17]. When HAVI-Fluc-MAVS was co-overexpressed with Rluc-MAVS in HEK293 cells, the percentage of Rluc on the beads compared with the cell lysate was 0.13%, which is at least 6-fold higher than that of three negative controls (P<0.05) and higher than the positive PPI cutoff value of 3-fold [6] (Fig. 6A). These data suggest that HAVI-Fluc-MAVS interacted with Rluc-MAVS when they were co-expressed, confirming the dimerization of MAVS. Moreover, the activity ratio of Rluc/Fluc of Rluc-MAVS and HAVI-Fluc-MAVS on the beads was approximately 0.28∶1(Fig. 6A), providing a relative amount of interacting HAVI-Fluc-MAVS and Rluc-MAVS. These results indicate that the DLR-PD assay is able to quantitatively measure the PPIs of membranous protein.


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 dimerization and inhibition by HCV NS3/4A in HEK293 cells.(A) Evaluation of the HAVI-Fluc-MAVS association with Rluc-MAVS using the DLR-PD assay. HEK293 cells were co-transfected with the indicated vectors (100 ng). The percentage of Rluc on the beads compared with the cell lysate was calculated by dividing the Rluc activity of Rluc-MAVS on the beads with the Rluc activity in the same amount of lysate used in the pull-down assays. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads. (B) HAVI-MAVS was cleaved by HCV NS3/4A and detected by Western blot when coexpressed in HEK293 cells. (C) Analysis of the interactions between HAVI-Fluc-MAVS and Rluc-MAVS inhibited by HCV NS3/4A using the DLR-PD assay. HEK293 cells were co-transfected with HAVI-Fluc-MAVS (100 ng), Rluc-MAVS (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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198426&req=5

pone-0026414-g006: Analysis of MAVS dimerization and inhibition by HCV NS3/4A in HEK293 cells.(A) Evaluation of the HAVI-Fluc-MAVS association with Rluc-MAVS using the DLR-PD assay. HEK293 cells were co-transfected with the indicated vectors (100 ng). The percentage of Rluc on the beads compared with the cell lysate was calculated by dividing the Rluc activity of Rluc-MAVS on the beads with the Rluc activity in the same amount of lysate used in the pull-down assays. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads. (B) HAVI-MAVS was cleaved by HCV NS3/4A and detected by Western blot when coexpressed in HEK293 cells. (C) Analysis of the interactions between HAVI-Fluc-MAVS and Rluc-MAVS inhibited by HCV NS3/4A using the DLR-PD assay. HEK293 cells were co-transfected with HAVI-Fluc-MAVS (100 ng), Rluc-MAVS (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.
Mentions: To determine whether the DLR-PD assay is applicable to the analysis of membranous protein interactions, MAVS dimerization was measured using the DLR-PD assay. MAVS is located on the outer membrane of mitochondria, and its self-association mediates antiviral innate immune signaling [16], [17]. When HAVI-Fluc-MAVS was co-overexpressed with Rluc-MAVS in HEK293 cells, the percentage of Rluc on the beads compared with the cell lysate was 0.13%, which is at least 6-fold higher than that of three negative controls (P<0.05) and higher than the positive PPI cutoff value of 3-fold [6] (Fig. 6A). These data suggest that HAVI-Fluc-MAVS interacted with Rluc-MAVS when they were co-expressed, confirming the dimerization of MAVS. Moreover, the activity ratio of Rluc/Fluc of Rluc-MAVS and HAVI-Fluc-MAVS on the beads was approximately 0.28∶1(Fig. 6A), providing a relative amount of interacting HAVI-Fluc-MAVS and Rluc-MAVS. These results indicate that the DLR-PD assay is able to quantitatively measure the PPIs of membranous protein.

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