Truly quantitative analysis of the firefly luciferase complementation assay ☆

摘要

Highlights ? Luciferase complementation assays (LCAs) detect protein interactions within cells. ? A mathematical model of the firefly LCA was constructed. ? The firefly LCA is qualitative but not quantitative. Abstract Luciferase complementation assays detect protein-protein interactions within living cells using bioluminescence. Since the first report using plant cells was published in 2007, over 100 peer-reviewed articles have been published describing the detection of protein-protein interactions within plant cells by the assays. The assays have also been used to analyze networks of protein-protein interactions in plants. Although the assays have a high dynamic range, they remain qualitative with respect to determining the affinities of interactions. In this article, we first summarize the luciferase complementation assays developed in the past years. We then describe the mechanism of the firefly luciferase complementation that is most widely used in plants, and the reason it is qualitative rather than quantitative using a mathematical model. Finally, we discuss possible procedures to quantitatively determine the affinity of a protein pair using the firefly luciferase complementation assay. Keywords Luciferase complementation ; Protein–protein interactions ; Quantitative assay ; Mathematical model ; Plant cells ; In vitro ; In cellulo ; In vivo prs.rt("abs_end"); 1. Luciferase complementation assays and their use for the network analysis of protein–protein interactions in animal cells Luciferase complementation assays (LCAs) detect protein–protein interactions within living cells using bioluminescence. In the assays, complementary DNA (cDNA) of luciferase is first split into the N- and C- terminal fragments and then fused to cDNAs of a protein pair of interest. A cell of interest is transformed or transfected with the resulting recombinant cDNAs so that a pair of the recombinant proteins is expressed within the cell. When the recombinant proteins interact with each other, the enzymatic activity of split luciferase is reconstituted. Compared with other assays that detect protein–protein interaction in living cells, these assays have a high dynamic range of interaction signals due to extremely low background signals in the samples [1] . Accordingly, LCAs are suitable to conduct high-throughput screening in which high degrees of differentiation between a positive and negative signal is required. The research group of Umezawa first published the principle of the LCA using luciferase obtained from fireflies ( Photinus pyralis ) in 2001 [2] . In the publication, insulin dose-dependent interactions of phosphorylated insulin receptor substrate 1 (IRS-1) and the N-terminal SH2 domain of PI 3-kinase in living Chinese hamster ovary (CHO) cells was described. As the assay was further modified, the research group of Jacob published the network analysis of protein–protein interactions in Human Embryonic Kidney 293 (HEK 293) cells with LCA using luciferase from copepod ( Gaussia princeps ) in 2012 [3] . The analyzed proteins are composed of a total of 2167 viral and human protein pairs. To identify the interacting protein pairs, they first benchmarked the assay using 100 randomly selected protein pairs for the negative result, and 143 protein pairs known to interact for the positive [4] . The detected luminescence was normalized by dividing the luminescence of a tested protein pair by the luminescence measured in control experiments. In the control experiments, they measured the luminescence emitted by the random interactions of the N- and C- fragments of luciferase. Frequency distributions for the normalized luminescence of positive and negative protein pairs were used to determine the threshold luminescence for an interacting protein pair. 2. Use of luciferase complementation assays for the network analysis of protein–protein interactions in plant cells We published the application of LCA in Arabidopsis protoplasts, for the first time, to detect the interaction of a histone protein pair using luciferase from sea pansy ( Renilla reniformis ) in 2007 [5] . We further published the network analysis of protein–protein interactions in Arabidopsis protoplasts using the same LCA in 2010 [6] . The analyzed proteins in the network are composed of 38 pairs of SNAREs (soluble N -ethylmaleimide-sensitive factor attachment protein). To identify the interacting protein pairs, we benchmarked the assay by comparing the previously published results of co-immunoprecipitation assays. Eleven negative protein pairs and 8 positive protein pairs identified using co-immunoprecipitation assays were compared to the LCA results [6] . The luminescence detected in the LCA was normalized by dividing the luminescence by the luminescence emitted by luciferase activity from the click beetle ( Pyrophorus plagiophthalamus ) that depends on the transformation efficiency of the cells. Distributions for the normalized luminescence values for the positiv