**4.3. S/B ratio in vitro**

The S/B ratio of FRET is rather low, but on the other hand, PCA shows a high S/B ratio and high sensitivity. The conventional FlimPIA described in Section 3.1 showed that the maximal S/B ratio was 2.5, which is generally lower than the S/B ratio of PCA [7]. However, the S/B ratio dramatically increased by the improvements described in Section 3.2-3.5 and was equal to or higher than the S/B ratio of PCA [6, 9, 10].

A Novel Protein-Protein Interaction Assay Based on the Functional Complementation of Mutant… http://dx.doi.org/10.5772/intechopen.75644 23

**Figure 9.** FKBP12-FRB association detected by FlimPIA in cultured cells. ©American Chemical Society.

**Figure 10.** Comparison of thermostability in vitro. (A) Probes (50 nM each) were preincubated at 37°C with or without equimolar rapamycin. The luminescent intensity was measured for 4 s after adding substrates (LH<sup>2</sup> and ATP). Left: FlimPIA, Right: Fluc-based PCA. Red: incubation for 0 min with rapamycin, Orange: 15 min with rapamycin, pink: 30 min with rapamycin, green: 0 min without rapamycin, light blue: 15 min without rapamycin, dark blue: 30 min without rapamycin. (n = 3) (B) Inactivation time course. Relative luminescent intensities at 4 s after reaction start were normalized at the value obtained with 0 min pre-incubation (n = 3). ©American Chemical Society.

#### **4.4. Sensitivity in vitro**

CoA to the mixture of FlimPIA (**Figure 8B**). In the presence of CoA, the maximum S/B ratio

Next, we optimized the concentration of ATP, as it was designed so that the *Km* value of the Acceptor for ATP was lower than that of the wild type to suppress the adenylation activity,

centration of ATP was 1 mM, and the maximal S/B ratio reached approximately 40, represent-

Finally, we had optimized the reaction conditions. As the increase of luminescence occurred as soon as substrates were added, a luminometer equipped with a stirrer was used to mix and react the substrates quickly (**Figure 8D**). The luminescence intensity increased quasi-linearly from 0.2 to 0.6 s after the reaction start and then reached a plateau. The maximal S/B ratio

Taken together, these improvements achieved a remarkably higher S/B ratio and sensitivity [6].

In this section, we describe the advantages and disadvantages of FlimPIA compared to the

To determine if FlimPIA is applicable in cellulo or in vivo, the FKBP-Donor and FRB-Acceptor were transiently expressed in cultured cells (**Figure 9**) [7]. The response was clearly observed in cells when rapamycin was added, and the luminescence intensity increased depending on

However, the maximal S/B ratio was less than 2.5, and the detectable range of the concentration of rapamycin was rather narrow. Although the S/B ratio of FRET is often as low as that of

The same Fluc derived from *P. pyralis* was applied to both Fluc-based PCA in vitro and FlimPIA. Then, the thermostability of probes was compared [10]. The probes of Fluc-based PCA (FKBP-C and FRB-N) and the probes of FlimPIA (FKBP-Donor and FRB-Acceptor) were preincubated with or without rapamycin at 37°C (**Figure 10A**). After 30 minutes, half of the luminescence signal was retained in FlimPIA, and on the other hand, the luminescence signal was almost completely diminished in PCA. The rate of the luminescence decay in FlimPIA

The S/B ratio of FRET is rather low, but on the other hand, PCA shows a high S/B ratio and high sensitivity. The conventional FlimPIA described in Section 3.1 showed that the maximal S/B ratio was 2.5, which is generally lower than the S/B ratio of PCA [7]. However, the S/B ratio dramatically increased by the improvements described in Section 3.2-3.5 and was equal

conventional PPI assay, FRET, and PCA, which are available in cellulo and in vitro.

FlimPIA in cells, PCA gives a high S/B ratio both in vitro and in cellulo.

was approximately one-fourth of the rate of the decay in PCA (**Figure 10B**).


reached 8, representing a twofold improvement, when 50 nM of each probe was used.

but the *Km* value of the Donor for ATP was maintained to provide LH<sup>2</sup>

reached more than 60 when 100 nM of each probe was used.

**4. Advantages and disadvantages of FlimPIA**

**4.1. FlimPIA in cells**

22 Protein-Protein Interaction Assays

the concentration of rapamycin.

**4.2. Stability of probes in vitro**

**4.3. S/B ratio in vitro**

to or higher than the S/B ratio of PCA [6, 9, 10].

ing a fivefold improvement, when 50 nM of each probe was used (**Figure 8C**).

The detectable limits of the concentration of rapamycin in Fluc-based PCA, the conventional FlimPIA, and the improved FlimPIA were compared, when 50 nM of each probe (FKBP-C and FRB-N, or FKBP-Donor and FRB-Acceptor) and rapamycin were used. The limits were 250 pM in Fluc-based PCA and 10 pM in FlimPIA using the K529Q/S440L mutant as the Acceptor [4, 9, 10]. The sensitivity of the improved FlimPIA was higher than the sensitivity of Fluc-based PCA.

were mixed with FRB-N, FRB-YPet, and FRB-Acceptors, respectively. As expected, the FRET signal was very weak when rapamycin was added to the mixture of FKBP12-Fn7-8-Cerulean and FRB-YPet, whereas the signal derived from the mixture of FKBP12-Cerulean and FRB-YPet was clearly observed (not shown). In the case of PCA using FKBP12-Fn7-8-C and FRB-N, the luminescence intensity was not significantly increased by the addition of rapamycin when the concentrations of the probes were moderate (100 nM each), while some response was observed with higher concentrations (750 nM) of each probe (not shown). However, the response of FlimPIA was clearly observed, even when 100 nM each of FKBP-Fn7-8-Donor and

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

25

We reported the development of Fluc-based PCA using purified probes for the first time. However, the stabilities of the probes were low due to the split forms. The problem might be

Furthermore, we developed a unique PPI assay, called FlimPIA, wherein the catalytic reaction of Fluc is divided into two half-reactions. FlimPIA has several advantages, especially in vitro.

This project was supported partly by SENTAN and SICORP, Japan Science and Technology agency, Japan; by JSPS KAKENHI Grant Numbers JP15H04191, JP17K06920, and JP24040072 from the Japan Society for the Promotion of Science, Japan; by Kikkoman Co.; by Dynamic Alliance for Open Innovation Bridging Human, Environment and Materials from MEXT,

Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute

overcome by using another enzyme with a highly stable structure.

Our next challenge is to improve the response in cellulo.

Japan; and by the 'Leave a Nest' Microtech-Nichion award.

The authors declare that there are no conflicts of interest.

Yuki Ohmuro-Matsuyama and Hiroshi Ueda\*

of Technology, Yokohama, Japan

\*Address all correspondence to: ueda@res.titech.ac.jp

FRB-Acceptor was used.

**Acknowledgements**

**Conflict of interest**

**Author details**

**5. Conclusions**

**Figure 11.** Detectable distance between the probes in vitro. (A) Scheme of the assays. A long (7 nm) Fn7-8 domain is inserted between a binding domain (FKBP12) and a probe. Signals with and without equimolar rapamycin were compared. (B) FRET using 40 nM each of FKBP-Fn7-8-Cerulean and the FRB-YPet as a probe pair. (C) Fluc-PCA using 100 nM each of FKBP-Fn7-8-C and FRB-N (n = 3). (D) FlimPIA using 100 nM each of FKBP-Fn7-8-Donor and FRB-Acceptor (left) (n = 3). ©American Chemical Society.

#### **4.5. Detection limit of dimension of interacting protein in vitro**

A fundamental limitation of FRET is that the detectable distance between the two probes is less than several nanometers, because the fluorescent signal is inversely proportional to the sixth power of the distance. A part of fibronectin type III, the seventh and eighth domains (Fn7-8), has a rigid structure with a 7 nm N-C terminal distance [10]. Ohashi et al. reported that a FRET signal using YPet and CyPet could not be observed by inserting Fn7-8 between the two fluorescent proteins [22]. The limit of the detectable distance between the two probes determines the detectable dimensions of the interacting protein.

Therefore, we compared the limit of the detectable distance between the probes in our assay. To examine this, Fn7-8 was inserted between FKBP12 and one of the probes (C-terminal domain for PCA, cerulean for FRET, and Donor for FlimPIA) (**Figure 11**). The large probes were mixed with FRB-N, FRB-YPet, and FRB-Acceptors, respectively. As expected, the FRET signal was very weak when rapamycin was added to the mixture of FKBP12-Fn7-8-Cerulean and FRB-YPet, whereas the signal derived from the mixture of FKBP12-Cerulean and FRB-YPet was clearly observed (not shown). In the case of PCA using FKBP12-Fn7-8-C and FRB-N, the luminescence intensity was not significantly increased by the addition of rapamycin when the concentrations of the probes were moderate (100 nM each), while some response was observed with higher concentrations (750 nM) of each probe (not shown). However, the response of FlimPIA was clearly observed, even when 100 nM each of FKBP-Fn7-8-Donor and FRB-Acceptor was used.
