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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 bFos-bJun interactions in HEK293 cells.(A) HAVI-Fluc-bJun associated with Rluc-bFos when co-expressed in HEK293 cells 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 percentage of Rluc on the beads compared with the lysate was calculated by dividing the Rluc activity 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) HAVI-Fluc-bJun association with Rluc-bFos when co-expressed in HEK293 cells using the biotinylated protein pull-down assay. The arrow indicates endogenous biotinylated proteins. PD, pull down; SA, streptavidin; WCL, whole cell lysate. (C) The Rluc/Fluc activity ratio of Rluc-Fluc and HAVI-Fluc-Rluc in HEK293 cell lysate (Left). The Rluc/Fluc activity ratio of HAVI-Fluc-Rluc in the cell lysate and on the beads (Right). HEK293 cells were transfected with Rluc-Fluc and HAVI-Fluc-Rluc plasmids (100 ng), and cells were collected at 48 h post-transfection.
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pone-0026414-g003: Analysis of bFos-bJun interactions in HEK293 cells.(A) HAVI-Fluc-bJun associated with Rluc-bFos when co-expressed in HEK293 cells 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 percentage of Rluc on the beads compared with the lysate was calculated by dividing the Rluc activity 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) HAVI-Fluc-bJun association with Rluc-bFos when co-expressed in HEK293 cells using the biotinylated protein pull-down assay. The arrow indicates endogenous biotinylated proteins. PD, pull down; SA, streptavidin; WCL, whole cell lysate. (C) The Rluc/Fluc activity ratio of Rluc-Fluc and HAVI-Fluc-Rluc in HEK293 cell lysate (Left). The Rluc/Fluc activity ratio of HAVI-Fluc-Rluc in the cell lysate and on the beads (Right). HEK293 cells were transfected with Rluc-Fluc and HAVI-Fluc-Rluc plasmids (100 ng), and cells were collected at 48 h post-transfection.

Mentions: Human Jun and Fos form a heterodimeric transcription factor. Notably, these proteins strongly interact in the cellular nucleus via their basic leucine zipper regions, making human Jun and Fos classic model protein pairs for the development of novel PPI assays [7], [11]. To establish DLR-PD assays for the analysis of PPIs, biotinylated Fluc tag and Rluc tag were fused to the basic leucine zipper regions of Jun and Fos, respectively, through a flexible linker 4x(G4S). When the DLR-PD assay was carried out to analyze the interaction between Jun and Fos, the percentage of Rluc on beads to the HAVI-Fluc-bJun and co-expressed Rluc-bFos lysate used to identify PPIs was approximately 4.0% (Fig. 3A), which is more than 80-fold the percentage for the other three negative controls (P<0.05) and well above the cutoff value of 3-fold for positive PPIs in the original LUMIER assays using Rluc-tagged protein [6]. These data suggest that HAVI-Fluc-bJun interacts with Rluc-bFos, which is consistent with the results obtained from the traditional pull-down assay (Fig. 3B). Because bJun and bFos interact in the cellular nucleus, the DLR-PD assay is suitable for the analysis of PPIs in the nucleus [11].


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 bFos-bJun interactions in HEK293 cells.(A) HAVI-Fluc-bJun associated with Rluc-bFos when co-expressed in HEK293 cells 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 percentage of Rluc on the beads compared with the lysate was calculated by dividing the Rluc activity 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) HAVI-Fluc-bJun association with Rluc-bFos when co-expressed in HEK293 cells using the biotinylated protein pull-down assay. The arrow indicates endogenous biotinylated proteins. PD, pull down; SA, streptavidin; WCL, whole cell lysate. (C) The Rluc/Fluc activity ratio of Rluc-Fluc and HAVI-Fluc-Rluc in HEK293 cell lysate (Left). The Rluc/Fluc activity ratio of HAVI-Fluc-Rluc in the cell lysate and on the beads (Right). HEK293 cells were transfected with Rluc-Fluc and HAVI-Fluc-Rluc plasmids (100 ng), and cells were collected at 48 h post-transfection.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198426&req=5

pone-0026414-g003: Analysis of bFos-bJun interactions in HEK293 cells.(A) HAVI-Fluc-bJun associated with Rluc-bFos when co-expressed in HEK293 cells 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 percentage of Rluc on the beads compared with the lysate was calculated by dividing the Rluc activity 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) HAVI-Fluc-bJun association with Rluc-bFos when co-expressed in HEK293 cells using the biotinylated protein pull-down assay. The arrow indicates endogenous biotinylated proteins. PD, pull down; SA, streptavidin; WCL, whole cell lysate. (C) The Rluc/Fluc activity ratio of Rluc-Fluc and HAVI-Fluc-Rluc in HEK293 cell lysate (Left). The Rluc/Fluc activity ratio of HAVI-Fluc-Rluc in the cell lysate and on the beads (Right). HEK293 cells were transfected with Rluc-Fluc and HAVI-Fluc-Rluc plasmids (100 ng), and cells were collected at 48 h post-transfection.
Mentions: Human Jun and Fos form a heterodimeric transcription factor. Notably, these proteins strongly interact in the cellular nucleus via their basic leucine zipper regions, making human Jun and Fos classic model protein pairs for the development of novel PPI assays [7], [11]. To establish DLR-PD assays for the analysis of PPIs, biotinylated Fluc tag and Rluc tag were fused to the basic leucine zipper regions of Jun and Fos, respectively, through a flexible linker 4x(G4S). When the DLR-PD assay was carried out to analyze the interaction between Jun and Fos, the percentage of Rluc on beads to the HAVI-Fluc-bJun and co-expressed Rluc-bFos lysate used to identify PPIs was approximately 4.0% (Fig. 3A), which is more than 80-fold the percentage for the other three negative controls (P<0.05) and well above the cutoff value of 3-fold for positive PPIs in the original LUMIER assays using Rluc-tagged protein [6]. These data suggest that HAVI-Fluc-bJun interacts with Rluc-bFos, which is consistent with the results obtained from the traditional pull-down assay (Fig. 3B). Because bJun and bFos interact in the cellular nucleus, the DLR-PD assay is suitable for the analysis of PPIs in the nucleus [11].

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