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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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Application of the DLR-PD assay to the domain mapping of the interaction of MAVS with TRAF3.(A) Western blot of HAVI-Fluc-tagged MAVS domains and Rluc-tagged TRAF3 in HEK293 cells. (B) Domain mapping of the interaction of MAVS with TRAF3 using the DLR-PD assay. HEK293 cells were co-transfected with Rluc-TRAF3, and the different domains of MAVS were fused with HAVI-Fluc vectors. MAVS CD, MAVS Card domain; MAVS PD, MAVS proline-rich domain; MAVS TM, MAVS transmembrane domain. 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-g008: Application of the DLR-PD assay to the domain mapping of the interaction of MAVS with TRAF3.(A) Western blot of HAVI-Fluc-tagged MAVS domains and Rluc-tagged TRAF3 in HEK293 cells. (B) Domain mapping of the interaction of MAVS with TRAF3 using the DLR-PD assay. HEK293 cells were co-transfected with Rluc-TRAF3, and the different domains of MAVS were fused with HAVI-Fluc vectors. MAVS CD, MAVS Card domain; MAVS PD, MAVS proline-rich domain; MAVS TM, MAVS transmembrane domain. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads.

Mentions: Protein interaction domain mapping is very important for studying PPIs. To determine whether the DLR-PD assay is suitable for domain mapping, the domain of MAVS, which is responsible for the interaction with TRAF3, was analyzed (Fig. 8A). The results indicated that the MAVS TM domain played a more important role than the proline-rich and CARD domains in MAVS-TRAF3 interactions (P<0.05) (Fig. 8B). However, the molecular integrity of MAVS was critical for MAVS and TRAF3 to interact, which is consistent with the result of the traditional pull-down assay (Fig. 7C).


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)

Application of the DLR-PD assay to the domain mapping of the interaction of MAVS with TRAF3.(A) Western blot of HAVI-Fluc-tagged MAVS domains and Rluc-tagged TRAF3 in HEK293 cells. (B) Domain mapping of the interaction of MAVS with TRAF3 using the DLR-PD assay. HEK293 cells were co-transfected with Rluc-TRAF3, and the different domains of MAVS were fused with HAVI-Fluc vectors. MAVS CD, MAVS Card domain; MAVS PD, MAVS proline-rich domain; MAVS TM, MAVS transmembrane domain. 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

Show All Figures
getmorefigures.php?uid=PMC3198426&req=5

pone-0026414-g008: Application of the DLR-PD assay to the domain mapping of the interaction of MAVS with TRAF3.(A) Western blot of HAVI-Fluc-tagged MAVS domains and Rluc-tagged TRAF3 in HEK293 cells. (B) Domain mapping of the interaction of MAVS with TRAF3 using the DLR-PD assay. HEK293 cells were co-transfected with Rluc-TRAF3, and the different domains of MAVS were fused with HAVI-Fluc vectors. MAVS CD, MAVS Card domain; MAVS PD, MAVS proline-rich domain; MAVS TM, MAVS transmembrane domain. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads.
Mentions: Protein interaction domain mapping is very important for studying PPIs. To determine whether the DLR-PD assay is suitable for domain mapping, the domain of MAVS, which is responsible for the interaction with TRAF3, was analyzed (Fig. 8A). The results indicated that the MAVS TM domain played a more important role than the proline-rich and CARD domains in MAVS-TRAF3 interactions (P<0.05) (Fig. 8B). However, the molecular integrity of MAVS was critical for MAVS and TRAF3 to interact, which is consistent with the result of the traditional pull-down assay (Fig. 7C).

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