**2. Demonstration of in vitro protein-fragment complementation assay using purified Fluc fragments**

Conventional PCA is used in vivo and in cultured cells (in cellulo). Although Porter et al. performed a Fluc-based PCA in vitro, the assay requires cell lysate, and the components in the lysate might affect the PPIs. We succeeded in developing a Fluc-based PCA in vitro using purified probes in a defined solution [4, 5].

For the Fluc-based PCA in vitro, a well-known interacting pair, FKBP12 (a 12 kD domain of FK506-binding protein) and FRB (FKBP-rapamycin-associated protein), was utilized. The association between these proteins depends on the presence of an antibiotic, rapamycin [11, 12]. Two pairs of split *Photinus pyralis* Fluc—the pair of the N-terminal domain (amino acids [aa] 1–437) and the C-terminal domain (aa 394–547) and the pair of the N-terminal domain (aa 1–398) and the same C-terminal domain (aa 394–547)—were selected in several split sites of Fluc [13], which worked well for in cellulo PCA. The gene encoding FKBP12 or FRB was fused to the 5' end of each domain, and the genes were inserted into the pET32 vector, which originally encodes thioredoxin (Trx), yielding four fusion protein genes, FKBP-N, FKBP-C, FRB-N, and FRB-C. These proteins were expressed in the soluble fraction of *E. coli* BL21(DE3) pLysS and purified by immobilized metal affinity chromatography (**Figure 1A**).

substrates, luciferin and ATP. On the other hand, the luminescence intensities of the mixture of the interacting pair (FKBP12 and FRB) without rapamycin and noninteracting pair were very low (**Figure 1C**). The signal and stability of the pair of the N-terminal domain (aa 1–437) and the C-terminal domain (aa 384–547) were higher than those of the other pair of N-terminal domain (aa 1–398) and the same C-terminal domain. When the first pair was used, the luminescence signal displayed rapamycin dose dependence, and the limit of detection was determined as 250 pM. These results clearly showed that the PPI could be detected with a high S/B

**Figure 1.** Detection via Fluc-based PCA using purified probes. (A–C) Detection of FKBP-FRB association. (A) Purified probes. Lane 1, FKBP-N; Lane 2, FRB-N; Lane 3, FRB-N; Lane 4, FRB-C. (B) PCA using the purified probes at 50 nM each, with/without equimolar rapamycin (n = 3). (C) Control experiments (n = 3). (D, E) Detection of p53-Mdm2 association. (D) PCA using the purified probes at 50 nM each (n = 3). (E) Inhibition of p53-Mdm2 interaction by Nutlin-3 (n = 3).

A Novel Protein-Protein Interaction Assay Based on the Functional Complementation of Mutant…

http://dx.doi.org/10.5772/intechopen.75644

13

Because the rapamycin-dependent FKBP-FRB association is very strong, another interacting pair, p53 and Mdm2, was investigated (**Figure 1D** and **E**) [14, 15]. p53 suppresses cell growth as a tumor suppressor. The oncoprotein Mdm2 binds to p53 and downregulates the function

ratio and high sensitivity using the purified probes.

©American Chemical Society

The two interacting pairs, FKBP-N and FRB-C, FKBP-C and FRB-N, were mixed, and rapamycin was added to the pair (**Figure 1B**). The luminescence intensities of the mixture of the interacting pairs and rapamycin were remarkably increased immediately after adding the two A Novel Protein-Protein Interaction Assay Based on the Functional Complementation of Mutant… http://dx.doi.org/10.5772/intechopen.75644 13

The conventional PPI assays, which are available both in vitro and in cellulo, are Förster/fluorescence resonance energy transfer (FRET)-based assays, bioluminescent resonance energy

For FRET-based assays, two fluorescent proteins or two fluorescent dyes are fused to proteins that interact with each other. When the interaction occurs, the two fluorescent proteins (dyes) are in close proximity, and then the energy transfer is induced, resulting in changes of the fluorescent intensities. In BRET assays, a bioluminescent enzyme and fluorescent protein (dye) are fused to proteins that interact with each other, and the energy is transferred from the bioluminescent enzyme to the fluorescent protein (dye). FRET- and BRET-based assays

For PCA, the enzyme or fluorescence is divided into two fragments. The split fragments are fused to interacting proteins. Upon interaction, the split fragments come close, and then the full length of the structure is reconstituted, resulting in the recovery of the enzyme activity or fluorescence. PCA in cells and lysates is a user-friendly method that gives a high signal/ background (S/B) ratio [3]. Moreover, we reported in vitro PCA using purified firefly luciferase (Fluc) fragments for the first time [4, 5]. The development of PCA is described in Section 2. Recently, we developed a novel PPI assay, named firefly luminescent intermediate-based proteinprotein interaction assay, FlimPIA [6–10]. FlimPIA utilizes the unique reaction of Fluc, which is divided into two half steps. We describe the principle of FlimPIA in Section 3.1 and the several improvements of FlimPIA in Sections 3.2–3.6. Then, the advantages and disadvantages of FlimPIA compared to another PPI assays such as the in vitro PCA are described in the final section.

**2. Demonstration of in vitro protein-fragment complementation** 

Conventional PCA is used in vivo and in cultured cells (in cellulo). Although Porter et al. performed a Fluc-based PCA in vitro, the assay requires cell lysate, and the components in the lysate might affect the PPIs. We succeeded in developing a Fluc-based PCA in vitro using

For the Fluc-based PCA in vitro, a well-known interacting pair, FKBP12 (a 12 kD domain of FK506-binding protein) and FRB (FKBP-rapamycin-associated protein), was utilized. The association between these proteins depends on the presence of an antibiotic, rapamycin [11, 12]. Two pairs of split *Photinus pyralis* Fluc—the pair of the N-terminal domain (amino acids [aa] 1–437) and the C-terminal domain (aa 394–547) and the pair of the N-terminal domain (aa 1–398) and the same C-terminal domain (aa 394–547)—were selected in several split sites of Fluc [13], which worked well for in cellulo PCA. The gene encoding FKBP12 or FRB was fused to the 5' end of each domain, and the genes were inserted into the pET32 vector, which originally encodes thioredoxin (Trx), yielding four fusion protein genes, FKBP-N, FKBP-C, FRB-N, and FRB-C. These proteins were expressed in the soluble fraction of *E. coli* BL21(DE3) pLysS and purified by immobilized metal affinity chromatography (**Figure 1A**). The two interacting pairs, FKBP-N and FRB-C, FKBP-C and FRB-N, were mixed, and rapamycin was added to the pair (**Figure 1B**). The luminescence intensities of the mixture of the interacting pairs and rapamycin were remarkably increased immediately after adding the two

transfer (BRET) assays, and protein-fragment complementation assays (PCAs).

are the most common and sophisticated methods.

12 Protein-Protein Interaction Assays

**assay using purified Fluc fragments**

purified probes in a defined solution [4, 5].

**Figure 1.** Detection via Fluc-based PCA using purified probes. (A–C) Detection of FKBP-FRB association. (A) Purified probes. Lane 1, FKBP-N; Lane 2, FRB-N; Lane 3, FRB-N; Lane 4, FRB-C. (B) PCA using the purified probes at 50 nM each, with/without equimolar rapamycin (n = 3). (C) Control experiments (n = 3). (D, E) Detection of p53-Mdm2 association. (D) PCA using the purified probes at 50 nM each (n = 3). (E) Inhibition of p53-Mdm2 interaction by Nutlin-3 (n = 3). ©American Chemical Society

substrates, luciferin and ATP. On the other hand, the luminescence intensities of the mixture of the interacting pair (FKBP12 and FRB) without rapamycin and noninteracting pair were very low (**Figure 1C**). The signal and stability of the pair of the N-terminal domain (aa 1–437) and the C-terminal domain (aa 384–547) were higher than those of the other pair of N-terminal domain (aa 1–398) and the same C-terminal domain. When the first pair was used, the luminescence signal displayed rapamycin dose dependence, and the limit of detection was determined as 250 pM. These results clearly showed that the PPI could be detected with a high S/B ratio and high sensitivity using the purified probes.

Because the rapamycin-dependent FKBP-FRB association is very strong, another interacting pair, p53 and Mdm2, was investigated (**Figure 1D** and **E**) [14, 15]. p53 suppresses cell growth as a tumor suppressor. The oncoprotein Mdm2 binds to p53 and downregulates the function of p53 in certain cancer cells. In the assay of p53-Mdm2 interaction using p53-C and Mdm2-N, the signal intensity and S/B ratio rose with higher concentrations of the probes of the interacting pair. To investigate the reversibility of the PCA, an inhibitor of the p53-Mdm2 interaction, Nutlin-3, was added to the mixture of p53-C and Mdm2-N. The luminescence intensity decreased depending on the concentration of Nutlin-3.

The invitro PCA opens the way to study PPIs of cytotoxic proteins, which is impossible to perform in cells. Furthermore, the possibility that the cellular components affect PPIs can be excluded.
