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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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Feasibility of the DLR-PD assay.(A) Scheme for biotinylating the Fluc fusion protein by BirA biotin ligase and Rluc fusion protein in Fig. 1. The sequence of 6xHis and 15-aa Avi tag fused to the N-terminus of Fluc is shown. The asterisk indicates the lysine residue that is specifically biotinylated by BirA. The sequence of the Flag tag fused to the C-terminus of Rluc is shown. MCS, multiple clone sites; Pcmv, human cytomegalovirus (CMV) immediate-early gene promoter; IRES, internal ribosomal entry site. (B) Western blot analysis for the expression of biotinylated Fluc in HEK293 cells transfected with either a HAVI-Fluc or Fluc expression vector (Left). Biotinylated Fluc was detected by Western blot after a pull-down assay was performed on the cell lysate of HEK293 cells transfected with HAVI-Fluc and Fluc (Right). A three-fold addition of the cell lysate and SA-PMPs (100 µl) were used in the pull-down assay. The protein was eluted by boiling the beads after binding. The same membrane was probed with either anti-Fluc antibodies or streptavidin conjugated horseradish peroxidase (SA-HRP). (C) The bead depletion efficiency of Fluc indicates the depleted bead Fluc activity ratio compared with the lysate. This value was calculated by dividing depleted Fluc activity of the beads by the Fluc activity of the same amount of lysate used for the pull down. HEK293 cells were transfected with HAVI-Fluc or Fluc expression vectors. A pull-down assay was performed using SA-PMPs, and the Fluc activity of the lysate and the activity of the remaining Fluc in the lysate after pull down were measured. The depleted Fluc activity of the beads was equal to the Fluc activity of lysate minus the activity of the remaining Fluc of the lysate after the binding to the beads. (D) Percentage of Fluc on the beads compared with the lysate after pull down using SA-PMPs. This value was calculated by dividing Fluc activity measured on the beads by the Fluc activity in the same amount of lysate used for pull down. HEK293 cells were transfected with HAVI-Fluc or Fluc expression vectors. The pull-down assay was performed using SA-PMPs, and the Fluc activity on the beads and in the cell lysate was measured. (E) Schematic diagram of Rluc-Fluc and HAVI-Fluc-Rluc fusion expression constructs (Upper). Western blots of assayed protein expression in HEK293 cells transfected with HAVI-Fluc, HAVI-Rluc, HAVI-Fluc-Rluc, Fluc, Rluc-Fluc and Rluc-Flag expression vectors. The thick arrow indicates the endogenous biotinylated proteins (Down). SA-HRP, streptavidin conjugated horseradish peroxidase. (F) HEK293 cells were transfected or co-transfected with the indicated expression vectors. The pull-down assay was performed using SA-PMPs, and the Fluc and Rluc activity on the beads and in the cell lysate was measured. Beads/lysate indicates that the Fluc or Rluc activity ratio of the beads to the lysate. This value was calculated by dividing the Fluc (or Rluc) activity measured on the beads by the Fluc (or Rluc) activity in the same amount of lysate for the pull down.
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pone-0026414-g002: Feasibility of the DLR-PD assay.(A) Scheme for biotinylating the Fluc fusion protein by BirA biotin ligase and Rluc fusion protein in Fig. 1. The sequence of 6xHis and 15-aa Avi tag fused to the N-terminus of Fluc is shown. The asterisk indicates the lysine residue that is specifically biotinylated by BirA. The sequence of the Flag tag fused to the C-terminus of Rluc is shown. MCS, multiple clone sites; Pcmv, human cytomegalovirus (CMV) immediate-early gene promoter; IRES, internal ribosomal entry site. (B) Western blot analysis for the expression of biotinylated Fluc in HEK293 cells transfected with either a HAVI-Fluc or Fluc expression vector (Left). Biotinylated Fluc was detected by Western blot after a pull-down assay was performed on the cell lysate of HEK293 cells transfected with HAVI-Fluc and Fluc (Right). A three-fold addition of the cell lysate and SA-PMPs (100 µl) were used in the pull-down assay. The protein was eluted by boiling the beads after binding. The same membrane was probed with either anti-Fluc antibodies or streptavidin conjugated horseradish peroxidase (SA-HRP). (C) The bead depletion efficiency of Fluc indicates the depleted bead Fluc activity ratio compared with the lysate. This value was calculated by dividing depleted Fluc activity of the beads by the Fluc activity of the same amount of lysate used for the pull down. HEK293 cells were transfected with HAVI-Fluc or Fluc expression vectors. A pull-down assay was performed using SA-PMPs, and the Fluc activity of the lysate and the activity of the remaining Fluc in the lysate after pull down were measured. The depleted Fluc activity of the beads was equal to the Fluc activity of lysate minus the activity of the remaining Fluc of the lysate after the binding to the beads. (D) Percentage of Fluc on the beads compared with the lysate after pull down using SA-PMPs. This value was calculated by dividing Fluc activity measured on the beads by the Fluc activity in the same amount of lysate used for pull down. HEK293 cells were transfected with HAVI-Fluc or Fluc expression vectors. The pull-down assay was performed using SA-PMPs, and the Fluc activity on the beads and in the cell lysate was measured. (E) Schematic diagram of Rluc-Fluc and HAVI-Fluc-Rluc fusion expression constructs (Upper). Western blots of assayed protein expression in HEK293 cells transfected with HAVI-Fluc, HAVI-Rluc, HAVI-Fluc-Rluc, Fluc, Rluc-Fluc and Rluc-Flag expression vectors. The thick arrow indicates the endogenous biotinylated proteins (Down). SA-HRP, streptavidin conjugated horseradish peroxidase. (F) HEK293 cells were transfected or co-transfected with the indicated expression vectors. The pull-down assay was performed using SA-PMPs, and the Fluc and Rluc activity on the beads and in the cell lysate was measured. Beads/lysate indicates that the Fluc or Rluc activity ratio of the beads to the lysate. This value was calculated by dividing the Fluc (or Rluc) activity measured on the beads by the Fluc (or Rluc) activity in the same amount of lysate for the pull down.

Mentions: To obtain biotinylated Fluc for the DLR-PD assay, Fluc with an HAVI tag sequence containing both 6 x His and Avi tags was coexpressed with bacterial biotin ligase enzyme BirA to covalently attach biotin to a specific lysine residue of the Avi Tag [10] (Fig. 2A). After the expression of HAVI-tagged Fluc in HEK293 cells, the biotinylation of this protein was determined by both Western blot and pull-down assays. As shown in Fig. 2B, HAVI-tagged Fluc was biotinylated, whereas Fluc was not. There was a small amount of unbiotinylated Fluc on the pull-down beads, representing nonspecific binding. The activity of the remaining Fluc in the cell lysate was quantitatively measured using the DLR assay after purification with Streptavidin MagneSphere Paramagnetic Particles (SA-PMPs). The Fluc activity of biotinylated HAVI-Fluc and nonbiotinylated Fluc were approximately 75% and 25% depleted, respectively (Fig. 2C). These data suggest that at least 50% of HAVI-Fluc was specifically bound to the beads if the nonspecific binding of HAVI-Fluc and Fluc are equal. However, when Fluc activity was directly measured on beads, only approximately 3.5% of the Fluc activity of HAVI-Fluc was detected on the beads (Fig. 2D). Notably, the nonspecific binding of unbiotinylated Fluc was significantly reduced after washing when Fluc activity was directly measured on beads (Fig. 2D). Therefore, Fluc activity was directly measured on beads instead of using the supernatant in subsequent experiments. These results suggest that biotinylated HAVI-Fluc fusion proteins can bind specifically to SA-PMPs beads. Importantly, the Fluc activity of the binding proteins can be quantitatively determined. To attenuate the effect of transfection variations on the amount of Fluc or Rluc bound to beads, the percentage of Fluc or Rluc activity of beads to cell lysate was used in all the experiments, which equals Fluc or Rluc activity on the beads divided by the Fluc or Rluc activity of the same amount of cell lysate used in the pull-down assays.


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)

Feasibility of the DLR-PD assay.(A) Scheme for biotinylating the Fluc fusion protein by BirA biotin ligase and Rluc fusion protein in Fig. 1. The sequence of 6xHis and 15-aa Avi tag fused to the N-terminus of Fluc is shown. The asterisk indicates the lysine residue that is specifically biotinylated by BirA. The sequence of the Flag tag fused to the C-terminus of Rluc is shown. MCS, multiple clone sites; Pcmv, human cytomegalovirus (CMV) immediate-early gene promoter; IRES, internal ribosomal entry site. (B) Western blot analysis for the expression of biotinylated Fluc in HEK293 cells transfected with either a HAVI-Fluc or Fluc expression vector (Left). Biotinylated Fluc was detected by Western blot after a pull-down assay was performed on the cell lysate of HEK293 cells transfected with HAVI-Fluc and Fluc (Right). A three-fold addition of the cell lysate and SA-PMPs (100 µl) were used in the pull-down assay. The protein was eluted by boiling the beads after binding. The same membrane was probed with either anti-Fluc antibodies or streptavidin conjugated horseradish peroxidase (SA-HRP). (C) The bead depletion efficiency of Fluc indicates the depleted bead Fluc activity ratio compared with the lysate. This value was calculated by dividing depleted Fluc activity of the beads by the Fluc activity of the same amount of lysate used for the pull down. HEK293 cells were transfected with HAVI-Fluc or Fluc expression vectors. A pull-down assay was performed using SA-PMPs, and the Fluc activity of the lysate and the activity of the remaining Fluc in the lysate after pull down were measured. The depleted Fluc activity of the beads was equal to the Fluc activity of lysate minus the activity of the remaining Fluc of the lysate after the binding to the beads. (D) Percentage of Fluc on the beads compared with the lysate after pull down using SA-PMPs. This value was calculated by dividing Fluc activity measured on the beads by the Fluc activity in the same amount of lysate used for pull down. HEK293 cells were transfected with HAVI-Fluc or Fluc expression vectors. The pull-down assay was performed using SA-PMPs, and the Fluc activity on the beads and in the cell lysate was measured. (E) Schematic diagram of Rluc-Fluc and HAVI-Fluc-Rluc fusion expression constructs (Upper). Western blots of assayed protein expression in HEK293 cells transfected with HAVI-Fluc, HAVI-Rluc, HAVI-Fluc-Rluc, Fluc, Rluc-Fluc and Rluc-Flag expression vectors. The thick arrow indicates the endogenous biotinylated proteins (Down). SA-HRP, streptavidin conjugated horseradish peroxidase. (F) HEK293 cells were transfected or co-transfected with the indicated expression vectors. The pull-down assay was performed using SA-PMPs, and the Fluc and Rluc activity on the beads and in the cell lysate was measured. Beads/lysate indicates that the Fluc or Rluc activity ratio of the beads to the lysate. This value was calculated by dividing the Fluc (or Rluc) activity measured on the beads by the Fluc (or Rluc) activity in the same amount of lysate for the pull down.
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Related In: Results  -  Collection

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pone-0026414-g002: Feasibility of the DLR-PD assay.(A) Scheme for biotinylating the Fluc fusion protein by BirA biotin ligase and Rluc fusion protein in Fig. 1. The sequence of 6xHis and 15-aa Avi tag fused to the N-terminus of Fluc is shown. The asterisk indicates the lysine residue that is specifically biotinylated by BirA. The sequence of the Flag tag fused to the C-terminus of Rluc is shown. MCS, multiple clone sites; Pcmv, human cytomegalovirus (CMV) immediate-early gene promoter; IRES, internal ribosomal entry site. (B) Western blot analysis for the expression of biotinylated Fluc in HEK293 cells transfected with either a HAVI-Fluc or Fluc expression vector (Left). Biotinylated Fluc was detected by Western blot after a pull-down assay was performed on the cell lysate of HEK293 cells transfected with HAVI-Fluc and Fluc (Right). A three-fold addition of the cell lysate and SA-PMPs (100 µl) were used in the pull-down assay. The protein was eluted by boiling the beads after binding. The same membrane was probed with either anti-Fluc antibodies or streptavidin conjugated horseradish peroxidase (SA-HRP). (C) The bead depletion efficiency of Fluc indicates the depleted bead Fluc activity ratio compared with the lysate. This value was calculated by dividing depleted Fluc activity of the beads by the Fluc activity of the same amount of lysate used for the pull down. HEK293 cells were transfected with HAVI-Fluc or Fluc expression vectors. A pull-down assay was performed using SA-PMPs, and the Fluc activity of the lysate and the activity of the remaining Fluc in the lysate after pull down were measured. The depleted Fluc activity of the beads was equal to the Fluc activity of lysate minus the activity of the remaining Fluc of the lysate after the binding to the beads. (D) Percentage of Fluc on the beads compared with the lysate after pull down using SA-PMPs. This value was calculated by dividing Fluc activity measured on the beads by the Fluc activity in the same amount of lysate used for pull down. HEK293 cells were transfected with HAVI-Fluc or Fluc expression vectors. The pull-down assay was performed using SA-PMPs, and the Fluc activity on the beads and in the cell lysate was measured. (E) Schematic diagram of Rluc-Fluc and HAVI-Fluc-Rluc fusion expression constructs (Upper). Western blots of assayed protein expression in HEK293 cells transfected with HAVI-Fluc, HAVI-Rluc, HAVI-Fluc-Rluc, Fluc, Rluc-Fluc and Rluc-Flag expression vectors. The thick arrow indicates the endogenous biotinylated proteins (Down). SA-HRP, streptavidin conjugated horseradish peroxidase. (F) HEK293 cells were transfected or co-transfected with the indicated expression vectors. The pull-down assay was performed using SA-PMPs, and the Fluc and Rluc activity on the beads and in the cell lysate was measured. Beads/lysate indicates that the Fluc or Rluc activity ratio of the beads to the lysate. This value was calculated by dividing the Fluc (or Rluc) activity measured on the beads by the Fluc (or Rluc) activity in the same amount of lysate for the pull down.
Mentions: To obtain biotinylated Fluc for the DLR-PD assay, Fluc with an HAVI tag sequence containing both 6 x His and Avi tags was coexpressed with bacterial biotin ligase enzyme BirA to covalently attach biotin to a specific lysine residue of the Avi Tag [10] (Fig. 2A). After the expression of HAVI-tagged Fluc in HEK293 cells, the biotinylation of this protein was determined by both Western blot and pull-down assays. As shown in Fig. 2B, HAVI-tagged Fluc was biotinylated, whereas Fluc was not. There was a small amount of unbiotinylated Fluc on the pull-down beads, representing nonspecific binding. The activity of the remaining Fluc in the cell lysate was quantitatively measured using the DLR assay after purification with Streptavidin MagneSphere Paramagnetic Particles (SA-PMPs). The Fluc activity of biotinylated HAVI-Fluc and nonbiotinylated Fluc were approximately 75% and 25% depleted, respectively (Fig. 2C). These data suggest that at least 50% of HAVI-Fluc was specifically bound to the beads if the nonspecific binding of HAVI-Fluc and Fluc are equal. However, when Fluc activity was directly measured on beads, only approximately 3.5% of the Fluc activity of HAVI-Fluc was detected on the beads (Fig. 2D). Notably, the nonspecific binding of unbiotinylated Fluc was significantly reduced after washing when Fluc activity was directly measured on beads (Fig. 2D). Therefore, Fluc activity was directly measured on beads instead of using the supernatant in subsequent experiments. These results suggest that biotinylated HAVI-Fluc fusion proteins can bind specifically to SA-PMPs beads. Importantly, the Fluc activity of the binding proteins can be quantitatively determined. To attenuate the effect of transfection variations on the amount of Fluc or Rluc bound to beads, the percentage of Fluc or Rluc activity of beads to cell lysate was used in all the experiments, which equals Fluc or Rluc activity on the beads divided by the Fluc or Rluc activity of the same amount of cell lysate used in the pull-down assays.

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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