A BRET-Based Homogeneous Insulin Assay Using Interacting

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A BRET-Based Homogeneous Insulin Assay Using Interacting Domains in the Primary Binding Site of the Insulin Receptor Hajime Shigeto,†,‡ Takeshi Ikeda,‡ Akio Kuroda,‡ and Hisakage Funabashi*,† †

Institute for Sustainable Sciences and Development, Hiroshima University, Higashihiroshima, Hiroshima 739-8511, Japan Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashihiroshima, Hiroshima 739-8530, Japan

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S Supporting Information *

ABSTRACT: A new homogeneous insulin assay requiring no chemical modification of an insulin recognition domain, which can be applied to continuous monitoring of the time-dependent cellular response in vitro, was developed. The carboxy-terminal α-chain (αCT) segment and first leucine-richrepeat (L1) domain in the primary binding site on the insulin receptor were genetically fused with a bioluminescent protein (Nanoluc, Nluc) and a fluorescent protein (yellow fluorescent protein, YPet) to produce the insulinsensing probe proteins Nluc−αCT and L1−YPet. The BRET signal was observed on simple mixing of insulin with these protein probes, in a so-called homogeneous assay. The BRET signal was proportional to the insulin concentration, and the lower detection limit was 0.8 μM. Time-dependent insulin secretion from drug-stimulated MIN6 cells was also successfully monitored continuously with the probe proteins. This BRET-based homogeneous insulin assay method is thus expected to be applicable to drug development by highthroughput screening. nsulin is secreted from pancreatic β-cells to control blood glucose level. Generally, insulin secretion is induced by high concentrations of glucose, such as that after a meal, and cells receiving the insulin begin to increase their glucose uptake, resulting in a decreased blood glucose level. In several cases of diabetes mellitus, this system of blood glucose level control via insulin does not function properly. Control of insulin secretion is accordingly an important goal of diabetes mellitus drug development. Sulfonylurea and its derivatives, among the bestknown drugs for type I diabetes mellitus, have upregulating effects on insulin secretion. However, long-term stimulation of pancreatic β-cells exerts oscillating or downregulating effects on insulin secretion in vitro.1−4 It is thus important to evaluate drug effects on time-dependent cellular response in insulin secretion. Conventionally, time-dependent insulin secretion from cultured β-cells is monitored by enzyme-linked immunosorbent assay (ELISA) with periodic sample collections and substitution of fresh medium.5 ELISA requires washing steps followed by signal development steps and thus offers limited time resolution; therefore, it should not be regarded as suitable for high-throughput screening (HTS). Other assay methods that do not require periodic sample collection or a washing step-free detection have also been reported. For example, a microfluidic device with fluorescent dye-modified insulin and fluorescent dye-modified antibody6−8 in a competitive immune assay format allows insulin detection without periodic sample collection, and fluorescence resonance energy transfer (FRET)-based analysis with fluorescent tag-modified antiinsulin antibodies9,10 and electrochemical analysis with anti-

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© XXXX American Chemical Society

insulin antibody or insulin binding aptamer-modified electrode11−13 enable washing-step-free detection. Although these methods are promising for continuous monitoring and HTS of time-dependent cellular response, the necessary complex chemical modifications of antibodies or aptamers as insulin recognition domains may result in low product yields. Insulin receptor activation state monitoring systems employing a genetically engineered insulin receptor have been developed.14−16 The Renilla luciferase (Rluc)-fused insulin receptor and enhanced yellow fluorescent protein (EYFP)fused insulin receptor were coexpressed in a mammalian cell. An insulin receptor forms a disulfide-linked homodimer, and thus the genetically fused insulin receptors also form the dimer with a certain probability. This dimerized insulin receptor alters its structure in response to insulin binding, resulting in a reduced distance between Rluc and EYFP that are fused for localization inside the cell membrane. The bioluminescence resonance energy transfer (BRET) signal between these reporter proteins parallels the insulin induced autophosphorylation, reflecting the activation state of the receptor, and it is proportional to the insulin concentration. In these studies, an in vitro system was also developed that employed insulin receptors partially purified by wheat germ lectin columns. This insulin-detection system has great potential to become one free of periodic sample collection and washing steps, without chemical modification of an insulin recognition domain Received: October 31, 2014 Accepted: February 6, 2015

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DOI: 10.1021/ac504063x Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

expression vector, in which Nluc and YPet were expressed in a fused form with a triple repeat of Gly-Gly-Gly-Gly-Ser [(GGGGS)3] as a linker, was designed. Synthesized oligos encoding (GGGGS)3 were first inserted into pcDNA3.1/mycHis B. The gene for Nluc and that for YPet were introduced into the N and C termini of the linker sequence, respectively. To construct a vector producing Nluc−αCT with a polyhistidine (His) tag, the synthesized DNA oligos coding αCT were first added to the C terminus of Nluc. Then the gene for Nluc−αCT was transferred to pcDNA3.1/myc-His A (Life Technologies), followed by the insertion of synthesized DNA oligos encoding the Ig κ-chain leader sequence21 in the N terminus. The L1−YPet with His-tag expression vector was constructed by the insertion of an L1 domain cloned from mouse embryonic stem cells (EB3,22,23 Riken, Japan) into the N terminus of the YPet gene, followed by the insertion of the Ig κ-chain leader sequence into the N terminus of the L1−YPet structural gene. For producing Nluc and YPet with a His tag as a control, synthesized DNA oligos encoding Ig κ-chain leader sequence were introduced into the N terminus of previously cloned genes on the pcDNA3.1/myc-His B vector. The structures of Nluc−(GGGGS)3−YPet, Nluc−αCT, and L1− YPet are depicted in Figure S1, Supporting Information. BRET Assay for Nluc−(GGGGS)3−YPet. CHO-K124 cells (Riken, Japan) were transfected with the Nluc−(GGGGS)3− YPet expression vector using Lipofectamine LTX (Life Technologies) following the manufacturer’s instructions. Two days after transfection, the BRET assay was performed with cellular lysate. First, Nano-Glo Luciferase Assay Buffer (Promega) was added to lyse the cells. The Nano-Glo Luciferase Assay Substrate (Promega) was then added to the lysate solution, and the luminescence spectrum from 400−600 nm was measured with a multimode microplate reader (Spectra-Max M5, Molecular Devices Japan, Tokyo, Japan). Protein Expression and Purification. Nluc−αCT, L1− YPet, Nluc, and YPet were expressed with an Expi293 Expression System (Life Technologies) following the manufacturer’s protocol. Seven days after transfection with the corresponding protein expression vectors, the supernatant of the cell culture medium was collected. The expression of the proteins in the medium was confirmed by SDS-PAGE and Western blotting (Figure S2a,b). The protein purification procedure was conducted using His-tag affinity. First, the collected supernatant was dialyzed against the His-tag affinity chromatography buffer (20 mM sodium phosphate, 500 mM sodium chloride, 5 mM imidazole, pH 7.4). Then the dialyzed sample was applied to a His-Trap FF column (GE Healthcare), and the target protein was eluted by imidazole gradation. The eluted protein was then dialyzed against either a Tris-HCl buffer (25 mM Tris-HCl, 137 mM sodium chloride, pH 8.0) or a KREBS-Ringer buffer (1.5 mM calcium chloride, 0.5 mM magnesium chloride, 4.5 mM potassium chloride, 120 mM sodium chloride, 0.7 mM sodium phosphate dibasic, 1.5 mM sodium phosphate monobasic, 15 mM sodium bicarbonate, pH 7.4). The status of the purified proteins was confirmed by SDSPAGE (Figure S3, Supporting Information), and the proteins were used in following experiments donated after measurement of concentration with a Micro BCA Kit (Thermo Scientific). Functional Analysis of Purified Proteins. The function of the purified proteins as an insulin-detecting probe was confirmed by luminescence and fluorescence analysis with recombinant insulin (Life Technologies), recombinant insulinlike growth factor-I (IGF-I) (Sigma-Aldrich), C-Peptide

such as on an antibody. However, because of the difficulty in further purification and mass production of membrane-bound proteins and the probability of formation of undesired combinations of receptor domains, applying this system for continuous monitoring of cellular response by mixing with a living cell culture is difficult. A new insulin-detecting method without chemical modification that can be applied to continuous monitoring and HTS of time-dependent cellular response is desirable. To address the problem, in this study, we focused on the structural relationship between insulin and insulin receptor. The carboxy-terminal αchain (αCT) segment binds to insulin, displacing the B-chain carboxy-terminal β-strand away from the insulin core structure. The residues of the exposed B-chain then contact the first leucine-rich-repeat (L1) domain in the primary binding site on the insulin receptor,17,18 suggesting the possibility of forming a complex with only the αCT segment, L1 domain, and insulin. We accordingly hypothesized that insulin could be detected in a homogeneous assay without a washing step, with a bioluminescent protein and fluorescent protein-fused insulininteracting domain as a sensing probe. These probe proteins produce a BRET signal between the bioluminescent protein and the fluorescent protein only when they form a complex with the insulin molecule (Figure 1). In addition, because these

Figure 1. Principle of the BRET-based homogeneous insulin assay. The interacting domains in the primary binding site of the insulin receptor, the carboxy-terminal α-chain (αCT) segment and first leucine-rich-repeat (L1) domain, were fused with bioluminescent proteins, Nanoluc (Nluc) and a fluorescent protein (YPet), respectively, to create the insulin sensing probe proteins, Nluc−αCT and L1−YPet. These probe proteins produced a BRET signal between Nluc and YPet only when they form a complex with the insulin molecule.

probe proteins are composed of relatively simple domains consisting of insulin-interacting domains and reporter proteins, it is expected to be possible to obtain large amounts of purified proteins independently and thus to use them in any desired concentration in a living cell culture. Here we report the BRET-based insulin assay with an αCTfused bioluminescent protein (Nanoluc; Nluc;19 Nluc−αCT) and an L1 domain-fused yellow fluorescent protein (YPet;20 L1−YPet) that is applicable to continuous monitoring of timedependent insulin secretion from cultured cells.

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EXPERIMENTAL SECTION Vector Construction. The details of the procedures used to construct protein expression vectors are described in Supporting Information. Briefly, the gene for Nluc from pNL 1.1 [Nluc] (Promega) and the gene for YPet from untargeted Aurora B FRET sensor Kif2 substrate (Addgene plasmid 45215) were inserted into pcDNA3.1/myc-His B (Life Technologies), respectively. A Nluc−(GGGGS)3−YPet protein B


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Analytical Chemistry (Yanaihara Institute, Shizuoka, Japan), and bovine serum albumin (BSA) (Nacalai Tesque, Kyoto, Japan). The spectra of luminescence with various combinations of purified proteins in a total volume of 95 μL were measured with a multimode microplate reader, with an integration time setting of 50 ms after the addition of 5 μL of Nano-Glo Luciferase Assay Substrate. BRET signal was evaluated by the measurement with a 50 ms integration time setting of luminescence intensities at 445 and 528 nm after the addition of 1 μL of Nano-Glo Luciferase Assay Substrate in a total volume of 100 μL. The BRET unit was calculated following the formula previously reported25 with modifications: [(emission at 528 nm) − (emission at 445 nm) × Cf)]/(emission at 445 nm), where Cf denotes (emission at 528 nm)/(emission at 445 nm) for a mixture of 0.5 μM Nluc−αCT and 100 μM L1−YPet, which is measured under the same experimental conditions. Continuous Monitoring of Time-Dependent Insulin Secretion from Cultured Mouse Pancreatic β-Cells. The mouse insulinoma pancreatic β-cell line, MIN6 cells,26,27 was kindly provided by Prof. Jun-ichi Miyazaki (Osaka University). MIN6 cells were maintained in Dulbecco’s modified Eagle’s medium containing 25 mM glucose (Life Technologies) with 11% (v/v) heat-inactivated fetal bovine serum (Thermo Scientific), 0.1 mM of 2-mercaptoethanol (Wako, Osaka, Japan), and 100 units/mL of penicillin and 100 μg/mL of streptomycin (Life Technologies) in a 5% CO2 atmosphere at 37 °C. For continuous monitoring of insulin secretion from drug-stimulated MIN6 cells, cells were collected and washed with KREBS-Ringer buffer. The harvested cells were inoculated into a 96-well microplate at 1 × 107 cells/well in a total volume of 50 μL. Nluc−αCT and L1−YPet were then added to the cellular solution to a final concentration of 0.5 μM and 100 μM, respectively. Nano-Glo Luciferase Assay Substrate (2.5 μL) mixed with cell-stimulating drugs, namely glucose (0, 5, 10, 15, 25 mM final), tolbutamide (3 μM final; Wako, Osaka, Japan), or diazoxide (10 μM final; Sigma-Aldrich) with glucose (25 mM final), was added and briefly pipetted. The BRET signal was immediately evaluated by measurement of bioluminescence at 445 and 528 nm with a 1000 ms integration time setting. The BRET unit was calculated as described above.

Figure 2. Luminescence and fluorescence properties of purified probe proteins. (a) Luminescence spectra of Nluc−αCT and Nluc measured with 5 μM of each protein, followed by the addition of 5 μL of NanoGlo Luciferase Assay Substrate. (b) Absorption spectra of L1−YPet and YPet measured with 5 μM of each protein. (c) Emission spectra of L1−YPet and YPet measured with 5 μM of each protein with 480 nm excitation.

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RESULTS AND DISCUSSION Characterization of Probe Proteins as a Bioluminescent Protein and a Fluorescent Protein. The basic properties of each probe protein as a bioluminescent protein and a fluorescent protein were characterized. The comparison of the bioluminescence spectrum between Nluc and Nluc−αCT revealed a shift of the emission peak from 458 to 445 nm (Figure 2a). This shift may have been caused by the structural distortion of Nluc fused with the αCT segment. Although it was difficult to compare the bioluminescent catalytic activities between them because of the impurity of Nluc alone (Figure S3, Supporting Information), we roughly estimated the quantum yield of Nluc−αCT. It was about 54% compared to the quantum yield of Nluc (see Supporting Information for the detailed calculation), suggesting Nluc−αCT as a potential bioluminescent protein. Next the fluorescence behavior of L1−YPet was also characterized. The 515 nm peak value of the absorption spectrum of L1−YPet was lower than that of YPet alone (Figure 2b). Accordingly, the peak of emission spectrum of L1−YPet was also weaker than that of YPet alone, as shown in Figure 2c.

Although detailed comparison of quantum yields was also difficult because of the impurity of YPet (Figure S3), we roughly estimated the quantum yield of L1−YPet, and it was about 0.20 (see Supporting Information for details). It was clear that fusion with the 35 kDa of the L1 domain has a strong impact on the fluorescence property of the fused YPet part. However, at least we showed that L1−YPet retains the function of a fluorescent protein. BRET-Based Homogeneous Insulin Assay with Purified Probe Proteins. First, the feasibility of BRET over the distance between the αCT segment and L1 domain forming a complex with the insulin molecule was confirmed. The distance between the αCT segment and the L1 domain in the complex was estimated as 5 nm by the crystal structure analysis previously reported.18 We accordingly linked Nluc and YPet with a triple-repeated GGGGS linker whose estimated length is C


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leading to lower BRET signals. However, the addition of a high, even 100-fold higher, concentration of insulin barely reduced the BRET signal (Figure 3b). Therefore, we concluded that the probe proteins form the complex by the same mechanism as that occurring in the insulin receptor. This mechanism indicates that Nluc−αCT is acting as the first limitation on final complex formation, and thus we considered that it would not be necessary to use the same amount of L1−YPet, whose binding constant is assumed to be different. The combination of 0.5 μM Nluc−αCT and 100 μM L1−YPet showed the highest BRET efficiency, obtained by a simple calculation as (emission at 528 nm)/(emission at 445 nm) and reached a plateau for 10 μM insulin detection (Figure 4a). In this combination, we consider that almost all Nluc−αCT−insulin “double” complexes are captured by a sufficient amount of L1−YPet to form the Nluc−αCT, L1−YPet, and insulin “triple” complex, and thus the BRET signal reached a plateau. The normalized spectra obtained with the best combination of probe proteins are

5 nm. The luminescence spectrum measured in a crude sample expressed by CHO-K1 cells indicates a clear BRET signal (Figure S4, Supporting Information), confirming the feasibility of the BRET assay with the probe proteins forming the complex in 5 nm. The luminescent spectra were then measured for the combination of purified Nluc−αCT and L1−YPet at the same molecular ratio. The raw spectra are shown in Figure 3a,

Figure 3. Luminescence spectra of an equal-ratio mixture consisting of Nluc−αCT and L1−YPet with different concentrations of insulin. (a) Luminescence spectra and (b) luminescence spectra normalized by the luminescence intensity at 445 nm. Each probe protein (5 μM) and insulin were mixed in a Tris-HCl buffer (pH 8.0) and incubated for 12 h at room temperature. The luminescence spectra were measured with the addition of 5 μL of Nano-Glo Luciferase Assay Substrate.

and the normalized spectra to make the comparison of the BRET efficiency as a sensing signal easy are depicted in Figure 3b. The emission peak (445 nm) attributed to the luminescence from Nluc−αCT (Figure 2a) decreased, and the intensity of 528 nm increased in response to insulin concentration (Figure 3a). In a BRET system, the emission energy of the luminescent donor is transferred to the acceptor, increasing the fluorescent emission. Therefore, the emission increase observed at around 528 nm can be regarded as a fluorescence emission of L1−Ypet (Figure 2c) resulting from BRET. The observation of BRET signal confirmed the formation of a complex of Nluc−αCT, L1−YPet, and insulin. It has been reported that engaged insulin in the primary binding site of the insulin receptor first binds to the αCT segment and changes its structure to be able to interact with the L1 domain. The probe proteins also appeared to form the complex by the same mechanism. If the probe proteins independently bind to an insulin molecule, the excess amount of insulin leaves a lower opportunity for the formation of the Nluc−αCT, L1−YPet, and insulin “triple” complex but increases the possibility of forming an Nluc−αCT−insulin “double” complex or a L1−YPet−insulin “double” complex,

Figure 4. BRET response with different concentrations of L1-YPet. (a) Luminescence at 445 and 528 nm was detected with 0.5 μM of Nluc−αCT, 10 μM of insulin, and different concentrations of L1− YPet in a KREBS-Ringer buffer. Purified probe proteins and insulin were mixed and incubated for 30 min at room temperature. The BRET efficiency was calculated as (emission at 528 nm)/(emission at 445 nm). The results of the experiment are shown as means ± standard deviation of three replicates. (b) The normalized spectra obtained with the best combination of probe proteins (0.5 μM Nluc−αCT and 100 μM L1−YPet). Each protein was mixed in a KREBS-Ringer buffer and incubated for 30 min at room temperature. The luminescence spectra were measured with the addition of 5 μL of Nano-Glo Luciferase Assay Substrate. The luminescence spectra were normalized by the intensity at 445 nm. D


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protein. The lower concentration of each protein also exhibited no BRET signal as shown in Figure S5, Supporting Information. Although, to the best of our knowledge, the actual mechanism of the activation of insulin receptor by IGF-I has not been elucidated, our result suggests that this does not depend on the αCT segment and L1 domain recognition. No signals in the case of C-peptide and BSA confirmed the specificity of the BRET signal, and thus we concluded that the probe proteins can be applied for the specific monitoring of insulin secretion from drug-stimulated pancreatic β-cells. We also emphasize that all measurements were performed with simple mixing of all ingredients without washing steps, as a so-called homogeneous assay, and thus this method can be applied to the continuous monitoring of cellular insulinsecreting response by the addition of purified probe proteins into the culture medium. Continuous Monitoring of Insulin Secretion from Drug-Stimulated MIN6 Cells. Finally, the BRET-based homogeneous insulin assay with the purified probe proteins was applied to continuous monitoring of insulin secretion from drug-stimulated MIN6 cells. The BRET signal after the drug stimulation was monitored continuously using the best combination of probe proteins optimized in Figure 4a (Figure 6). When the cells were stimulated by a high concentration of

shown in Figure 4b. The comparison of the spectrum from 0.5 μM of Nluc−αCT only and the one from 0.5 μM of Nluc−αCT + 100 μM of L1−YPet revealed the possibility of a concentration-induced collision-based BRET. Therefore, we calculated all BRET unit values for further experiments with the Cf defined as (emission at 528 nm)/(emission at 445 nm) for 0.5 μM of Nluc−αCT + 100 μM of L1−YPet. Nevertheless, the BRET efficiencies measured with the best combination of probe proteins were dramatically improved compared to those measured with the same molar ratio of the probe proteins (Figure 3b) and were clearly proportional to the insulin concentration, as shown in Figure 5a The lower detection limit of 3.29 standard deviations from the average of the blank value (0 μM insulin)28 was 0.8 μM.

Figure 6. Continuous monitoring of BRET signal measured with drugstimulated MIN6 cells. Nluc−αCT and L1−YPet were added to MIN6 cells in a KREBS-Ringer buffer to final concentrations of 0.5 μM and 100 μM, respectively. Nano-Glo Luciferase Assay Substrate (2.5 μL) mixed with cell-stimulating drugs, namely glucose (0, 5, 10, 15, 25 mM final), tolbutamide (3 μM final), or diazoxide (10 μM final) with glucose (25 mM final), was added. The luminescence was immediately measured at 445 and 528 nm with a 1000 ms integration time setting followed by measurement every 10 s for 60 min. The BRET unit was calculated as described in Experimental Section.

Figure 5. Luminescence response of the BRET-based homogeneous assay with the optimized combination of probe proteins. (a) Dose− response of the BRET-based homogeneous insulin assay. (b) The BRET signals measured with 0 μM insulin, 100 μM insulin, 100 μM IGF-I, 100 μM C-Peptide, and 100 μM BSA. Nluc−αCT (0.5 μM), L1−YPet (100 μM), and the protein of interest (each concentration) were mixed in a KREBS-Ringer buffer and incubated for 30 min at room temperature. The luminescence was measured with the addition of 1 μL of Nano-Glo Luciferase Assay Substrate. The results in the figures are shown as means ± standard deviation of three replicates.

glucose (10 mM, 15 mM, 25 mM), the BRET signals increased at approximately 10 min. It is the basic function of MIN6 cells to secrete insulin in response to a high concentration of glucose, and thus the increase of the BRET signal can be regarded as the result of an increase in insulin concentration due to cellular secretion. It has been reported that the postprandial blood glucose level, in case of murine, reaches 15 mM, while the fasting blood glucose level typically exhibits about 5 mM.31 In fact, no BRET signal was observed in case of

To discuss the specificity of the BRET signal, the following three proteins were measured with probe proteins: insulin-like growth factor-I (IGF-I), another protein example that activates the insulin receptor;14,16,29 C-peptide, a peptide that is cleaved from pro-insulin in a secretary vesicle and secreted from pancreatic β-cells together with matured insulin;30 bovine serum albumin (BSA) as a negative control. As shown in Figure 5b, no clear signals were observed even with 100 μM of each E



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ACKNOWLEDGMENTS We thank Prof. Jun-ichi Miyazaki (Osaka University) for the kind donation of MIN6 cells. This work was partially supported by JSPS KAKENHI grant numbers 23686119 and 26289314.

0 mM and 5 mM glucose stimulation. Therefore, we believe that the BRET signal response indeed reflects the functional role of β-cells. The BRET signal in response to a high concentration of glucose decreased after reaching the maximum. We roughly measured the insulin concentrations at 0, 10, and 60 min under the 25 mM glucose stimulation with ELISA. The insulin concentration in the buffer indeed decreased (Figure S6, Supporting Information), confirming that the decrease of the BRET signal reflects the dynamic insulin concentration reduction assumably attributed to cellular insulin uptake.32 Stimulation with tolbutamide, a derivative of sulfonylurea that is known to be an insulin secretion-upregulating drug,33 also resulted in a BRET signal increase at approximately 10 min, supporting the feasibility of continuous monitoring of drug-stimulated insulin secretion. With stimulation by a high concentration of glucose mixed with diazoxide that reduces insulin secretion,34 almost no BRET signal was observed. This result also supports the feasibility of evaluating the downregulating effect of a drug on insulin secretion. Under the defined conditions, the BRET signal reached a plateau approximately 20 min after mixture of the probe proteins with insulin (Figure S7, Supporting Information). This response speed may cause a delay of the BRET signal increase in response to drug stimulation. For this reason, this method cannot be regarded as a real-time monitoring method. However, these results demonstrate the potential of the BRET-based insulin assay with Nluc−αCT and L1−YPet for continuous monitoring of time-dependent insulin secretion from cultured cells without periodic sample collection or washing steps.

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CONCLUSION In this study, a new insulin-detection method without chemical modification of a target recognition domain was developed. The insulin-interacting domains in the primary binding site of the insulin receptor, the αCT segment and L1 domain, were genetically fused with a bioluminescent protein, Nluc, and a fluorescent protein, YPet. The BRET signal measured by simple mixture of insulin with these proteins as insulin-sensing probe proteins was proportional to the insulin concentration. Therefore, we conclude that probe proteins can be used to develop a BRET-based homogeneous insulin assay. In addition, time-dependent insulin secretion from drug-stimulated MIN6 cells was successfully monitored using the probe proteins without periodic sample collection or washing steps. This insulin assay method is thus expected to be applied to drug development by HTS. ASSOCIATED CONTENT

S Supporting Information *

Additional information and discussion as noted in the text. This material is available free of charge via the Internet at http:// pubs.acs.org.

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*Tel/Fax: +81-82-424-7893. E-mail: [email protected]. jp. Notes

The authors declare no competing financial interests. F


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A BRET-Based Homogeneous Insulin Assay Using Interacting

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