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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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Comparison of IRF3 dimerization using the DLR-PD assay and activation of the IRF3 regulatory element PRDIII-I induced by different molecules in HEK293 cells.(A) Schematic diagram of virus-activated innate antiviral signaling pathways. (B) Activation of the IRF3 regulatory element PRDIII-I induced by different expression vectors (control pCDNA3.1, GFP2-RIG-I, Halo-MDA5, eYFP-MAVS, Myc-TRAF3, Myc-TBK1 or Myc-IKKε) in HEK293 cells 24 h post-transfection. The IRF3 regulatory element PRDIII-I was assayed with 4xPRDIII-I-Fluc and the pRL-TK dual reporter. After being normalized by Rluc activity, Fluc activity from different cell lysates was compared with that of control pCDNA3.1-transfected cells. (C) Analysis of the association of HAVI-Fluc-IRF3 with Rluc-IRF3 induced by different molecules using the DLR-PD assay. HEK293 cells were co-transfected with HAVI-Fluc-IRF3 (20 ng), Rluc-IRF3 (10 ng) and different expression vectors (100 ng of control pCDNA3.1, GFP2-RIG-I, Halo-MDA5, eYFP-MAVS, Myc-TRAF3, Myc-TBK1 or Myc-IKKε). Subsequently, the DLR-PD assay was performed. 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-g005: Comparison of IRF3 dimerization using the DLR-PD assay and activation of the IRF3 regulatory element PRDIII-I induced by different molecules in HEK293 cells.(A) Schematic diagram of virus-activated innate antiviral signaling pathways. (B) Activation of the IRF3 regulatory element PRDIII-I induced by different expression vectors (control pCDNA3.1, GFP2-RIG-I, Halo-MDA5, eYFP-MAVS, Myc-TRAF3, Myc-TBK1 or Myc-IKKε) in HEK293 cells 24 h post-transfection. The IRF3 regulatory element PRDIII-I was assayed with 4xPRDIII-I-Fluc and the pRL-TK dual reporter. After being normalized by Rluc activity, Fluc activity from different cell lysates was compared with that of control pCDNA3.1-transfected cells. (C) Analysis of the association of HAVI-Fluc-IRF3 with Rluc-IRF3 induced by different molecules using the DLR-PD assay. HEK293 cells were co-transfected with HAVI-Fluc-IRF3 (20 ng), Rluc-IRF3 (10 ng) and different expression vectors (100 ng of control pCDNA3.1, GFP2-RIG-I, Halo-MDA5, eYFP-MAVS, Myc-TRAF3, Myc-TBK1 or Myc-IKKε). Subsequently, the DLR-PD assay was performed. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads.

Mentions: IRF3 dimerization is an important indicator of IRF3 activation. The assay to measure the activity of IRF3 regulatory element PRDIII-I regulated reporter induced by the over-expression of different molecules in the cytoplasmic antiviral signal pathway [13], [14] (Fig. 5A) indicated that MDA5, MAVS, TBK1, and IKKε induced approximately only a 2-fold IRF3 reporter activation in HEK293 cells (Fig. 5B). In contrast, using the established DLR-PD assay, the IRF3 dimerization ratio increased up to a thousand-fold when some molecules, such as IKKε, were overexpressed (Fig. 5C). Although the DLR-PD assay indicates IRF3 dimer formation and the 4xPRDIII-I luciferase activity reflects the activation of endogenous IRF3[15], the results of both assays are consistent when monitoring IRF3 activation.


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)

Comparison of IRF3 dimerization using the DLR-PD assay and activation of the IRF3 regulatory element PRDIII-I induced by different molecules in HEK293 cells.(A) Schematic diagram of virus-activated innate antiviral signaling pathways. (B) Activation of the IRF3 regulatory element PRDIII-I induced by different expression vectors (control pCDNA3.1, GFP2-RIG-I, Halo-MDA5, eYFP-MAVS, Myc-TRAF3, Myc-TBK1 or Myc-IKKε) in HEK293 cells 24 h post-transfection. The IRF3 regulatory element PRDIII-I was assayed with 4xPRDIII-I-Fluc and the pRL-TK dual reporter. After being normalized by Rluc activity, Fluc activity from different cell lysates was compared with that of control pCDNA3.1-transfected cells. (C) Analysis of the association of HAVI-Fluc-IRF3 with Rluc-IRF3 induced by different molecules using the DLR-PD assay. HEK293 cells were co-transfected with HAVI-Fluc-IRF3 (20 ng), Rluc-IRF3 (10 ng) and different expression vectors (100 ng of control pCDNA3.1, GFP2-RIG-I, Halo-MDA5, eYFP-MAVS, Myc-TRAF3, Myc-TBK1 or Myc-IKKε). Subsequently, the DLR-PD assay was performed. 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-g005: Comparison of IRF3 dimerization using the DLR-PD assay and activation of the IRF3 regulatory element PRDIII-I induced by different molecules in HEK293 cells.(A) Schematic diagram of virus-activated innate antiviral signaling pathways. (B) Activation of the IRF3 regulatory element PRDIII-I induced by different expression vectors (control pCDNA3.1, GFP2-RIG-I, Halo-MDA5, eYFP-MAVS, Myc-TRAF3, Myc-TBK1 or Myc-IKKε) in HEK293 cells 24 h post-transfection. The IRF3 regulatory element PRDIII-I was assayed with 4xPRDIII-I-Fluc and the pRL-TK dual reporter. After being normalized by Rluc activity, Fluc activity from different cell lysates was compared with that of control pCDNA3.1-transfected cells. (C) Analysis of the association of HAVI-Fluc-IRF3 with Rluc-IRF3 induced by different molecules using the DLR-PD assay. HEK293 cells were co-transfected with HAVI-Fluc-IRF3 (20 ng), Rluc-IRF3 (10 ng) and different expression vectors (100 ng of control pCDNA3.1, GFP2-RIG-I, Halo-MDA5, eYFP-MAVS, Myc-TRAF3, Myc-TBK1 or Myc-IKKε). Subsequently, the DLR-PD assay was performed. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads.
Mentions: IRF3 dimerization is an important indicator of IRF3 activation. The assay to measure the activity of IRF3 regulatory element PRDIII-I regulated reporter induced by the over-expression of different molecules in the cytoplasmic antiviral signal pathway [13], [14] (Fig. 5A) indicated that MDA5, MAVS, TBK1, and IKKε induced approximately only a 2-fold IRF3 reporter activation in HEK293 cells (Fig. 5B). In contrast, using the established DLR-PD assay, the IRF3 dimerization ratio increased up to a thousand-fold when some molecules, such as IKKε, were overexpressed (Fig. 5C). Although the DLR-PD assay indicates IRF3 dimer formation and the 4xPRDIII-I luciferase activity reflects the activation of endogenous IRF3[15], the results of both assays are consistent when monitoring IRF3 activation.

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