Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation

Abstract

In this study we developed an inducible synthetic renilla luciferase protein-fragment-assisted complementation-based bioluminescence assay to quantitatively measure real time protein-protein interactions in mammalian cells. We identified suitable sites to generate fragments of N and C portions of the protein that yield significant recovered activity through complementation. We validate complementation-based activation of split synthetic renilla luciferase protein driven by the interaction of two strongly interacting proteins, MyoD and Id, in five different cell lines utilizing transient transfection studies. The expression level of the system was also modulated by tumor necrosis factor alpha through NFkappaB-promoter/enhancer elements used to drive expression of the N portion of synthetic renilla luciferase reporter gene. This new system should help in studying protein-protein interactions and when used with other split reporters (e.g., split firefly luciferase) should help to monitor different components of an intracellular network.

Figure 1

Schematic diagram showing the protein-fragment-assisted complementation (PCA) strategy using split synthetic renilla luciferase (hrluc) to monitor protein–protein interactions. N-terminal portion of synthetic renilla luciferase is attached to protein X through the linker peptide (GGGGS)₂, and C-terminal portion of synthetic renilla luciferase is connected to protein Y through the linker (GGGGS)₂. Interaction of proteins X and Y recovers hrluc activity through protein complementation and produces light in the presence of the substrate coelenterazine and oxygen.

Figure 2

A. Schematic diagram showing different split points with nucleotide positions in 5′ to 3′ direction. The right-directed arrow (→) indicates the forward priming position and the left-directed arrow (←) indicates the reverse priming positions. The positive signs at nucleotide positions 669–670 and 687–688 indicate the split points restored activity during complementation with interacting proteins MyoD and Id. B. Amino acid (311) sequence of synthetic renilla luciferase protein with bolded amino acids showing the split sites. C. Diagram showing the plasmid constructs (i, ii) made for each split site with interacting proteins MyoD and ID under the control of the CMV promoter with linker (GGGGS)₂. (iii). The N-hrluc–Id construct with NFκB promoter/enhancer elements to modulate the system.

Figure 3

Protein–protein interaction mediated fragment-assisted complementation of the split hrluc system in transiently transfected 293T cells. The signal from cells cotransfected with both N-hrluc–Id and C-hrluc–MyoD shows significant recovered activity as compared to cells transfected with N-hrluc–Id alone and also significant recovered activity as compared to all other plasmids shown. The signal from cells transfected with C-hrluc–MyoD is not significantly different from mock-transfected cells. The error bar is the SEM of six samples.

Figure 4

A. Comparison of protein–protein interaction-mediated fragment-assisted complementation of split firefly luciferase, split synthetic renilla luciferase and native reporters of firefly luciferase, renilla luciferase, and synthetic renilla luciferase in transiently transfected 293T cells. The error bar is the SEM of six samples. B. The TNF-α-modulated protein–protein interaction-mediated fragment-assisted complementation of the split hrluc system in transiently transfected 293T cells. The error bar is the SEM of six samples.