Biosensor system for monitoring EPOR dimer formation induced by ligand, and target ligand detection method using same

Abstract

The present invention relates to a biosensor system for monitoring EPOR dimer formation by ligands and a target ligand detection method using same, wherein EPOR dimer formation by ligand binding can be rapidly and sensitively monitored using a fusion protein in which the EPOR ectodomain and a split fluorescent protein are fused. In addition, it is possible to monitor EPOR dimer formation by ligands and detect target ligands in vitro, and thus the complexity and duration of cell-based assays can be reduced, and high-speed screening can be easily applied. In addition, by measuring the reference fluorescence of a reference fluorescent protein together along with a signal fluorescence and comparing the reference fluorescence and signal fluorescence, accurate and quantitative analysis can be performed regardless of changes in measurement environment or variations in protein concentration.

Description

Biosensor system for monitoring ligand-induced EPOR receptor dimer formation and method for detecting target ligand using the same

[0001] The present invention relates to monitoring the dimer formation of EPOR receptors, and more specifically, to a biosensor system for monitoring the dimer formation of EPOR receptors induced by a ligand and a method for detecting a target ligand using the same.

[0002]

[0003] In vivo, intercellular or intracellular signal transduction occurs through the interaction of specific receptors and ligands that bind to them. This signal transduction process regulates almost every aspect of life phenomena, such as cell growth, differentiation, and metabolism, and abnormalities can lead to various diseases. Therefore, the technology to accurately monitor the binding of specific ligands to receptors and the subsequent receptor activation process is crucial in the fields of life science research and new drug development. Conventionally, various methods have been used to measure receptor activation. For example, it was common practice to measure the amount of phosphorylated proteins—the products of receptor activation—using biochemical assays such as ELISA (Enzyme-Linked Immunosorbent Assay) or Western Blot, or to analyze gene expression levels induced by downstream signaling pathways using PCR (Polymerase Chain Reaction). However, these methods generally had disadvantages, such as requiring cell destruction, being time-consuming, having limitations in quantitative analysis, and making it difficult to observe the initial events immediately following receptor-ligand binding in real time. In particular, many receptors initiate activation signals through dimerization, in which two receptor molecules bind to each other after ligand binding. For example, the erythropoietin receptor EPOR, which is essential for hematopoiesis, forms a dimer of EPOR molecules upon binding to the ligand erythropoietin EPO; this is a key step in initiating intracellular signal transduction. Directly monitoring this dimerization process is one of the most direct and accurate methods for evaluating the initial activation of receptors and the efficacy of ligands.Conventional dimerization monitoring techniques such as FRET (Forster Resonance Energy Transfer), BRET (Bioluminescence Resonance Energy Transfer), or Single-Molecule Tracking have been studied. However, since FRET and BRET require the use of two different fluorescent / luminescent proteins, the preparation process is complex, and sensitivity and quantification may be limited due to issues such as overlap between the emission wavelengths of each protein or interference from background signals. Furthermore, Single-Molecule Tracking has the disadvantage of requiring expensive equipment and advanced technology, making it difficult to apply to high-throughput screening. Additionally, for receptors with complex signaling pathways, such as the erythropoietin receptor (EPOR), measuring only the final activation steps, such as intracellular phosphorylation, may not reflect the initial ligand binding efficiency or the dynamics of dimer formation itself. Since the dimerization of EPOR is a critical step determining the biological activity of EPO, there is an urgent need for the development of novel biosensor systems and analytical methods capable of measuring this process rapidly, simply, and sensitively. In particular, a system capable of efficiently monitoring the dimerization of cell membrane proteins in an in vitro environment without the complexity of cell culture would be highly useful for screening potential activators or inhibitors. Therefore, it is necessary to develop innovative technologies that overcome the complexity, low sensitivity, and limitations of real-time monitoring of conventional techniques, enabling the direct, efficient, and sensitive measurement of receptor dimer formation induced by ligand binding.

[0004]

[0005] The present invention was carried out with the support of the following project.

[0006] [Project ID] 2460000771

[0007] [Assignment No.] KH141178

[0008] [Ministry Name] Ministry of Health and Welfare

[0009] [Name of Project Management (Specialized) Agency] Korea Health Industry Development Institute

[0010] [Research Project Name] Innovative Growth Skin Health Base Technology Development Project

[0011] [Project Title] Development of Novel Whitening Materials through Research on the Mechanism of Action of Erythropoietin on the Skin

[0012] [Name of Project Performing Organization] Daegu Gyeongbuk Institute of Science and Technology

[0013] [Research Period] 2024.01.01 ~ 2024.12.31

[0014]

[0015] The technical problem that the present invention aims to solve is to provide a biosensor system for monitoring the formation of EPOR receptor dimers induced by a ligand.

[0016] In addition, the technical problem that the present invention aims to solve is to provide a method for detecting a target ligand using the biosensor.

[0017]

[0018] The technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0019]

[0020] To achieve the above technical objective, one embodiment of the present invention provides a biosensor system for monitoring the dimerization of an EPOR receptor induced by a ligand, comprising: a first fusion protein in which a first ectodomain of an erythropoietin receptor (EPOR) and a first split fluorescent protein fragment are fused; and a second fusion protein in which a second erythropoietin receptor (EPOR) ectodomain and a second split fluorescent protein fragment are fused, wherein the first and second ectodomains become dimerized upon ligand binding.

[0021] In an embodiment of the present invention, the first and second split fluorescent protein fragments may be assembled upon dimerization of the first and second EPOR ectododomains to emit signal fluorescence.

[0022] In an embodiment of the present invention, the second split fluorescent protein fragment may have a reference fluorescent protein additionally connected thereto.

[0023] In an embodiment of the present invention, the split fluorescent protein fragment may be a first split green fluorescent protein (split-enhanced GFP, spEGFP) and a second split green fluorescent protein (split-enhanced GFP, spEGFP); or mCitrine and mTFP1.

[0024] In an embodiment of the present invention, the first split fluorescent protein fragment may be composed of the amino acid sequence of SEQ ID NO. 1, and the second split fluorescent protein fragment may be composed of the amino acid sequence of SEQ ID NO. 2 or 3.

[0025] In an embodiment of the present invention, the first split fluorescent protein fragment may be composed of the amino acid sequence of SEQ ID NO. 4, and the second split fluorescent protein fragment may be composed of the amino acid sequence of SEQ ID NO. 5.

[0026] In an embodiment of the present invention, the system may operate in vitro.

[0027] To achieve the above technical problem, another embodiment of the present invention provides a target ligand detection method using a biosensor system, comprising the steps of: mixing the first fusion protein and the second fusion protein with a sample containing or expected to contain a target ligand; and measuring signal fluorescence resulting from the assembly of the split fluorescent protein fragment in the mixture.

[0028] In an embodiment of the present invention, the detection method utilizes the biosensor system, and the measuring step may include the step of measuring the signal fluorescence according to the assembly of the split fluorescent protein fragment in the mixture and the reference fluorescence of the reference fluorescent protein.

[0029] In an embodiment of the present invention, the target ligand may be natural erythropoietin (EPO).

[0030]

[0031] The present invention relates to a biosensor system for monitoring the formation of EPOR receptor dimers induced by a ligand and a method for detecting a target ligand using the same, wherein the formation of EPOR dimers by ligand binding can be monitored rapidly and sensitively by using a fusion protein formed by fusing the ectodomain of EPOR with a split fluorescent protein.

[0032] In addition, since EPOR dimer formation by ligands can be monitored and target ligands detected in vitro, the complexity and time of cell-based analysis can be reduced, and it can be easily applied to high-speed screening.

[0033] In addition, by measuring the reference fluorescence of the reference fluorescent protein together and comparing it with the signal fluorescence, accurate and quantitative analysis can be performed regardless of changes in the measurement environment or fluctuations in protein concentration.

[0034]

[0035] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the composition of the invention described in the description or claims of the present invention.

[0036]

[0037] Figure 1 shows the design of a biosensor system using an spGFP assembly system.

[0038] Figure 2 briefly illustrates a strategy for cloning other receptors.

[0039] Figure 3 shows the amino acid sequence of a fusion protein expressed by cloning the gene encoding EPOR-mCitrine or EPOR-mTFP1 into the pET28b plasmid, where the amino acids of the EPOR ectodomain are shown in purple, mCitrine in yellow, and mTFP1 in cyan.

[0040] Figure 4 illustrates BiFC purification. a) Schematic diagram of the BiFC purification process. Purification was performed using various sequential chromatography methods to increase the purity of the protein samples. b) His-tag affinity chromatography purification profile (Left: EPOR_GFP_1-10, Right: EPOR_GFP_11_sfCherrry3C). c) Anion exchange chromatography purification profile (Left: EPOR_GFP_1-10, Right: EPOR_GFP_11_sfCherrry3C). d) SDS-PAGE profile of the purified BiFC. Each lane represents 1 / 2, 1 / 4, 1 / 8, and 1 / 16 of the stock sample (from left to right).

[0041] Figure 5 shows that the gap and ratio scores for various BiFC concentrations were calculated by detecting for 2 hours. The test was performed three times. 1 U is an analytical unit defined as the concentration at which EPOR_GFP_11_sfCherry3C emits a signal intensity of 1000 under dimerization analysis conditions. The gap score is calculated by subtracting the green signal from the red signal, and the ratio score is calculated by subtracting 1 from the value obtained by dividing the green signal from the red signal.

[0042] Figure 6 shows the Gap and Ratio scores for various BiFC concentrations collected at the end of the analysis. The experiment was performed three times and analyzed using one-way ANOVA and Turkey's multiple comparison test. In terms of Ratio scores, 'BiFC 1U EPO 1 μM' showed a significantly higher score (p < 0.001) compared to all other test groups. On the other hand, there was no significant difference compared to other test groups even with the same sample.

[0043] Figure 7 shows the Gap and Ratio scores of denatured EPO, calculated over 2 hours of detection. The experiment was performed three times. EPO was denatured by incubating at 95°C for 20 minutes. Denatured EPO (dEPO) showed dimerization activity that was 4-fold and 3-fold lower in the Gap and Ratio scores, respectively.

[0044] Figure 8 shows the structure of the modified BiFC. EPOR_GFP_1-10 (left) and GFP_11_sfCherry3C (right). Light blue: EPOR ectodomain, red: sfCherry3C, light green: spGFP-11 fragment, pale green: spGFP-1-10. This structure was visualized using the Discovery Studio visualization tool (Biovia).

[0045] Figure 9 shows the gap and ratio scores of the modified BiFC, calculated by detection for 2 hours. The samples were tested three times. The GFP_11_sfCherry3C group had gap and ratio scores 25 times and 3 times lower, respectively, compared to the control group. Due to the stronger red signal emitted at a concentration of 1 U, the gap score of GFP_11_sfCherry3C was relatively lower than that of the control group using EPOR_GFP_11_sfCherry3C.

[0046] Figures 10 and 11 show the results of cloning another fluorescent protein in a biosensor system according to an embodiment of the present invention.

[0047]

[0048] The present invention will be described in detail below.

[0049]

[0050] The present invention relates to a biosensor system for monitoring the dimer formation of EPOR receptors induced by a ligand.

[0051] The system of the present invention comprises a first fusion protein in which a first erythropoietin receptor (EPOR) ectodomain and a first split fluorescent protein fragment are fused; and a second fusion protein in which a second erythropoietin receptor (EPOR) ectodomain and a second split fluorescent protein fragment are fused, wherein the first and second ectodomains dimerize upon ligand binding.

[0052] The above EPOR ectodomain is a site that binds to a ligand such as extracellular erythropoietin (EPO), and may be the ectodomain sequence of natural EPOR or a modified sequence having an equivalent function. As long as the ectodomain maintains the ligand binding and dimer-forming properties of EPOR, cleaved or modified forms may also be utilized without restriction.

[0053] The above EPOR ectododomain includes sequences that are identical or complementary to each other and become dimerized upon ligand binding. Here, identical sequences include cases where a homodimer is formed upon ligand binding, and complementary sequences may include cases where a heterodimer is formed upon ligand binding. For example, it may include the amino acid sequence of SEQ ID NO. 6.

[0054] The first and second split fluorescent protein fragments may be assembled upon dimerization of the first and second EPOR ectododomains to emit signal fluorescence. Specifically, the fusion protein is designed to fuse the ectododomain, which is the ligand binding site of EPOR, with the split fluorescent protein fragment so that a fluorescent signal is generated only when a ligand is present and dimerization is induced, which is useful for directly and quantitatively monitoring the initial stages of EPOR activation.

[0055] The above-mentioned split fluorescent protein fragments do not exhibit fluorescence on their own, but they may have the characteristic of restoring fluorescence when assembled in close proximity to each other. Therefore, ligand detection may be possible based on the principle that when a target ligand binds to an ectodomain and induces dimerization, the split fluorescent protein fragments that come in close proximity are assembled to generate a fluorescent signal.

[0056] The second split fluorescent protein fragment may have a reference fluorescent protein additionally linked thereto. More specifically, the second fusion protein may have a second erythropoietin receptor (EPOR) ectododomain, the second split fluorescent protein fragment, and a reference fluorescent protein linked sequentially. The reference fluorescent protein provides a constant fluorescent signal regardless of dimerization caused by the binding of the target ligand, and can be used for the normalization of signal fluorescence. This can help obtain accurate and reliable ligand detection results regardless of deviations in sensor expression levels or quantitative variations in the sample.

[0057] The above-mentioned split fluorescent protein fragment may be prepared by splitting various fluorescent proteins commonly used in the industry, such as the GFP (green fluorescent protein), YFP (yellow fluorescent protein), CFP (blue-green fluorescent protein), and RFP (red fluorescent protein) series, and may, without limitation, split fragments such as spEGFP, mCitrine, mTFP1, Venus, and mCherry may be used. These may be suitable for implementing the principle in which fluorescence is restored by the reassembly of the split fragment through dimerization upon ligand binding.

[0058] The first and second split fluorescent protein fragments may be, for example, a first split green fluorescent protein (split-enhanced GFP, spEGFP) and a second split green fluorescent protein (split-enhanced GFP, spEGFP); or mCitrine and mTFP1.

[0059] For a specific example, the first split fluorescent protein fragment may be composed of the amino acid sequence of SEQ ID NO. 1, and the second split fluorescent protein fragment may be composed of the amino acid sequence of SEQ ID NO. 2 or 3. Additionally, the first split fluorescent protein fragment may be composed of the amino acid sequence of SEQ ID NO. 4, and the second split fluorescent protein fragment may be composed of the amino acid sequence of SEQ ID NO. 5.

[0060] The above system may operate in vitro. This means that the interaction and dimerization of ligands and receptors can be observed without using cells, which has technical significance in that it eliminates the complexity of cell culture and processing and can increase the speed of screening. In vitro, the purified first and second fusion proteins can be used to induce a reaction by mixing them with a sample expected to contain the target ligand.

[0061] The present invention relates to a method for detecting a target ligand using the biosensor system.

[0062] The method of the present invention comprises the steps of: mixing the first fusion protein and the second fusion protein with a sample that includes or is expected to include a target ligand; and measuring signal fluorescence according to the assembly of the split fluorescent protein fragment in the mixture.

[0063] Detailed explanations have been omitted for parts that overlap with the aforementioned content.

[0064] The step of mixing the first fusion protein and the second fusion protein with a sample containing or expected to contain a target ligand may be performed using means known by methods and conditions known in the art. The mixing concentration of the first and second fusion proteins may be determined by considering the intensity of signal fluorescence, the possibility of non-specific background signal generation, protein manufacturing costs, etc.

[0065] The above samples may include, without limitation, any type of sample that may contain a target ligand, such as biological samples (e.g., serum, plasma, urine, saliva, tissue extracts), environmental samples (e.g., water, soil extracts), or solutions prepared in a laboratory.

[0066] The step of measuring signal fluorescence resulting from the assembly of the split fluorescent protein fragment in the above mixture may involve quantifying the degree of dimerization according to the presence and concentration of the target ligand as fluorescence intensity. For example, this can be characterized by fluorescence measurement means commonly used in the art, such as a fluorometer, a fluorescence plate reader, or a fluorescence microscope. This characteristic may have technical significance in directly confirming that the split fluorescent protein fragment is reassembled and expresses fluorescence when dimerization of the ectodomain by the target ligand occurs.

[0067] The above detection method may, for example, determine that the sample contains the target ligand if the intensity of the signal fluorescence of the sample is stronger compared to a control sample that does not contain the target ligand. Additionally, if the intensity of the signal fluorescence of the sample is weaker compared to a control sample that does not contain the target ligand, or if there is no significant difference, the sample may be determined not to contain the target ligand. Furthermore, by comparing the intensity of the signal fluorescence of two samples that contain or are expected to contain the target ligand, it may be determined that the sample showing a stronger intensity contains the target ligand at a higher concentration.

[0068] When the above detection method utilizes a biosensor system including a reference fluorescent protein, the measuring step may include a step of measuring the signal fluorescence resulting from the assembly of the split fluorescent protein fragment in the mixture and the reference fluorescence of the reference fluorescent protein.

[0069] The above detection method may, for example, calculate a ratio or gap using the signal fluorescence and reference fluorescence and quantitatively determine the presence or concentration of a target ligand by comparing it with a preset reference value or standard curve.

[0070] Specifically, this may be a process of calculating the Signal / Reference Fluorescence Ratio by normalizing the signal fluorescence to the reference fluorescence, and determining the presence and concentration of the ligand by utilizing the fact that this ratio has a positive correlation with the concentration of the target ligand. This may have technical significance in that it improves detection accuracy by correcting for environmental factors or differences in sensor expression levels between samples by utilizing the ratio.

[0071] The above target ligand may be natural erythropoietin (EPO).

[0072] By using the biosensor system of the present invention, natural EPO and modified EPO can be clearly distinguished and detected. Furthermore, since natural EPO is the natural ligand of EPOR, the biosensor system of the present invention can be usefully applied for measuring the potency of EPO preparations or measuring the concentration of EPO in blood samples.

[0073]

[0074] Hereinafter, in order to specifically explain the present invention, it will be described in detail with reference to examples.

[0075]

[0076] Materials and Methods

[0077] 1. Design of a Biosensor System Using an SpGFP Assembly System

[0078] In the case of bimolecular fluorescence complementation (BiFC) using the spEGFP system, spEGFP1-10 were linked to the EPOR ectodomain molecule, and spEGFP11 was linked to the EPOR ectodomain and sfCherry3C. The EPOR ectodomain and spEGFP were connected by a short loop containing the DNA cleavage site of EcoRI, which was designed to allow for the exchange of the ectodomain portion with other desired target sites in the future. The size of the linker sequences connecting each domain was determined through structural prediction using Alphafold 2, which allowed for the calculation of appropriate linker lengths that do not interfere with the accurate folding of individual domains. The genes were fused to the pET28b(+) vector, and further transformation and cloning were performed in an E. coli system (Figs. 1 and 2).

[0079]

[0080] 2. Gene Synthesis

[0081] The plasmids used in this study and their corresponding maps are presented in Table 1 and Figure 1. Genes encoding EPOR-mCitrine or EPOR-mTFP1 were synthesized at Bionics (South Korea) and cloned into the pET28b plasmid. This plasmid contains an N-terminal hexahistidine tag sequence for nickel affinity purification. Genetic integrity was further evaluated using Sanger sequencing. The amino acid sequences of the DNA sequences were consistent with the initial design of the protein (Figure 3).

[0082]

[0083] Table 1. Strains and plasmids used in this studyE. coliStrainDH5αfor plasmid work, including mini-prepRosetta2 (DE3) pLysSfor protein overexpressionplasmidspET28bgene synthesis and overexpressionGenesmCitrineFPbase (https: / www.fpbase.org / protein / mcitrine / )mTFP1FPbase (https: / www.fpbase.org / protein / mtfp1 / )

[0084] 3. Target gene cloning

[0085] The receptor ectodomain of the biosensor system was replaced via vector plasmid cloning. The target gene was synthesized in Bionics and subcloned using In-Fusion® Snap Assembly Master Mix (TAKARA). Insertion primers were prepared according to the instructions in the provided kit and designed to overlap the upstream NdeI region of the vector plasmid pET28b(+) with the downstream EcoRI region of the target gene sequence. PCR was performed by applying the insertion primers to vectors containing various types of receptor ectodomains to generate the insertion fragments required for In-Fusion assembly. The vector plasmid was cleaved with restriction enzymes NdeI and EcoRI-HF (NEB) to obtain linear DNA of the target gene from which the receptor domain was excluded. The insertion primers and linear vector were fused to In-Fusion® Snap Assembly Master Mix, transformed into E. coli DH5α strains, and further screened in kanamycin LB medium. The vectors from the selected colonies were further collected using AccuPrep® Plasmid Mini.

[0086]

[0087] 4. Transformation, Culture, and Overexpression in E. coli Systems

[0088] The synthesized gene was transformed into eligible cells of the 'Rosetta2(DE3)' E. coli strain. Eligible cells were obtained using the 'Mix & Go! E.coli Transformation Kit and Buffer Set' (Zymo Research), and the subsequent transformation steps followed those provided by the manufacturer. After transformation, colonies were selected on LB agar plates containing 1 µg / mL kanamycin. The selected colonies were cultured overnight in 30 mL of kanamycin-LB medium and then inoculated into 1 mL of kanamycin-LB medium. When a concentration of 0.5% was reached, 1 mg of IPTG was added to initiate overexpression of the target gene. Soluble proteins were cultured overnight at 16 °C, while insoluble proteins were cultured overnight at 37 °C to improve yield.

[0089]

[0090] 5. Purification of target protein

[0091] After E. coli overexpression, the cells were centrifuged at 4,000 rpm. The pellet was lysed by adding 50 mM Tris-HCl (pH 8.0), 1 mM PMSF, and 2 mM DTT. Cell lysates were obtained by further sonicating the solvent, and separated by centrifugation at 13,000 rpm for 30 minutes. For soluble proteins, the supernatant was filtered through a 0.45 μm membrane and applied to an FPLC system. Conversely, for insoluble proteins, inclusion bodies were further separated from the pellet obtained from the cell lysates. Inclusion bodies were obtained by extracting insoluble cell debris using a detergent-containing buffer. The cell lysate pellet was first resuspended in a buffer containing 50 mM Tris-HCl (pH 8.0), 0.2 M NaCl, and 1% NP-40, and then centrifuged at 13,000 rpm for 30 minutes. The supernatant obtained from the previous step was removed, and the pellet was resuspended in a buffer solution containing 50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 0.5% Triton X-100, and 0.5% Tween-20. After final centrifugation at 13,000 rpm for 30 minutes, the supernatant of the sample was removed to separate the inclusion bodies. The inclusion bodies were extracted using 6 M urea and 50 mM Tris-HCl (pH 8.0) buffer, filtered through a 0.45 μm membrane to remove contaminants from the sample, and used for FPLC analysis.

[0092] The samples prepared in the previous step were purified using an FPLC system utilizing a chelation affinity column and an anion exchange column. For soluble proteins, the samples were first placed into a Hitrap Chelating column (Cytiva) and washed with 50 mM Tris-HCl buffer containing 50 mM imidazole, 0.5 M NaCl, 1 mM PMSF, and 2 mM DTT, followed by further elution with the same buffer containing a high concentration of imidazole (0.5 M). To increase the purity of the elution fraction, the samples were further purified using a Hitrap Q anion exchange column (Cytiva). To eliminate interference from NaCl ions contained in the elution buffer of the previous purification, the samples were dialyzed in 50 mM Tris-HCl (pH 8.0) containing 1 mM PMSF and 2 mM DTT. After dialysis, the sample was injected into a HitrapQ column and washed with 50 mM Tris-HCl buffer (pH 8.0) containing 1 mM PMSF and 2 mM DTT. The column was eluted sequentially by adding guanidine-HCl (300 mM) to the same buffer. Meanwhile, insoluble proteins underwent the same purification process by adding a 6 M urea buffer.

[0093] Both soluble and insoluble proteins were finally dialyzed in 50 mM Tris-HCl (pH 8.0) and applied to the analysis system (Fig. 4).

[0094]

[0095] 6. Normalization of the molar ratio of sensor molecules

[0096] To set the dimerization probabilities of EPOR1-10 and EPOR11 to be equal while excluding interference from trace impurities, the molar ratio was normalized by quantifying the bandwidth of the SDS-PAGE profile. The size of the target protein band was quantified using the computer program ImageJ. Samples were serially diluted to 1 / 2, 1 / 4, 1 / 8, and 1 / 16 and plotted as a linear function of the concentration-bandwidth graph. The molar ratio of each sample was calculated by comparing the slopes of the plotted linear function. The slope indicates that the amount of protein increased when the concentration changed twofold. The bandwidth graph is R 2 It was displayed carefully as the value can reach 0.99 or higher.

[0097] The standard for the molar ratio was established by defining 1 U as the amount of EPOR11 with a signal intensity of 1000 under fluorescence detection conditions, which corresponds to 1 μM of sensor molecules. Detection was performed using a TECAN Spark 10M, and the detection parameters were 'Gain 95, Green signal excitation 485 nm & emission 535 nm, Red signal excitation 555 nm & emission 625 nm, Z-position 20000 μm, integration time 80 μs, 30 flashes'. To improve the initial response rate, the temperature was controlled to be maintained at 37°C.

[0098]

[0099] 7. Detection of signal emission in biosensor systems

[0100] Biosensor activity was detected and quantified by calculating the relative values ​​of fluorescence intensity. Since the detector does not provide clear units, significance was measured by comparing signal intensities between test groups. The first criterion for evaluating the signal intensity of the test groups was the 'signal score,' obtained by dividing the green signal intensity by the red signal intensity. To investigate the effect of the ligand on spGFP assembly in more detail, 'gap scores' and 'ratio scores,' representing subtraction and division between the test and control groups, were introduced. The gap score is calculated as 'test group signal score' minus 'control group signal score' and indicates a positive linear correlation with the ligand's spGFP assembly effect. Conversely, the ratio score is calculated as 'test group signal score' / 'control group signal score' minus 1 and indicates a positive logistic correlation with the ligand's assembly effect. The protein content of the analysis system was calculated based on the final concentration under detection conditions. Each well contained a total of 100 μL of a mixture of BiFC protein, ligand, and buffer. Fluorescence detection was performed at 5-minute intervals to prevent photobleaching effects. The EPO ligand used in this study was purchased from peprotech (human recombinant EPO).

[0101]

[0102] The sequence used in the present invention is as follows.

[0103]

[0104] result

[0105] 1. Biosensor Characteristics

[0106] Several preliminary tests were performed before introducing the newly designed proteins into the experiment. Both EPOR_GFP_1-10 and EPOR_GFP_11_sfCherry3C were observed to form insoluble inclusion bodies upon overexpression. Additionally, EPOR_GFP_11_sfCherry3C contained residual protein fragments that were not removed even after sequential purification steps using His-tag affinity chromatography, anion exchange chromatography, and hydrophobic affinity chromatography. Furthermore, these proteins tended to aggregate easily at high concentrations under refrigerated conditions. Therefore, to maintain solubility during the purification process, the proteins were spread using 6M urea buffer.

[0107] In addition, the red signal of sfCherry3C showed a tendency for its intensity to gradually decrease when measured using a fluorescence detector, which is likely due to photofading caused by UV exposure. However, no signal attenuation was observed in the experimental group cultured under various temperature conditions during the same period.

[0108]

[0109] 2. Confirmation of biosensor activity

[0110] The BiFC-based biosensor system was tested by varying the receptor concentration while maintaining a constant ligand concentration (1 μM EPO) (Figs. 5 and 6). For the BiFC molecule, the concentration was measured using the previously defined analytical unit (U), which is equivalent to the amount of EPOR_GFP_11_sfCherry3C emitting a signal intensity of 1000 in the analytical system (1 U corresponds to approximately 1 μM of the receptor molecule). Receptor concentrations (1 U, 2 U, 4 U, 8 U, 16 U) directly influenced the observed Gap and Ratio scores. The experimental results showed an inverse relationship between receptor concentration and the Gap and Ratio scores. As the receptor concentration increased, the proportion of ligand-bound receptor complexes decreased, resulting in a weaker fluorescence signal. For example, the Gap score at 1 U was significantly higher than at 16 U, indicating that the spGFP fragment was assembled more effectively due to the higher ligand-to-receptor ratio. Conversely, as the receptor concentration increased, excess receptor molecules reduced the potential for ligand-induced receptor dimerization, thereby decreasing the observable fluorescence intensity.

[0111] The Gap score, representing the absolute fluorescence difference, decreased nearly linearly with increasing receptor concentration, highlighting sensitivity to changes in receptor availability. This trend suggests that the Gap score is a reliable indicator for detecting overall fluorescence activity when the receptor-to-ligand ratio is relatively balanced. Conversely, the Ratio score, which normalizes fluorescence change, flattened as receptor concentration increased, effectively capturing the system's saturation kinetics. This flattening phenomenon implies that while fluorescence intensity initially increases with receptor availability, the system eventually reaches a point where the ligand becomes a limiting factor, leading to signal saturation. These results indicate that the biosensor is robust over a wide range of receptor concentrations and provides valuable data even as the ligand-to-receptor ratio decreases.

[0112] Further analysis revealed that ratio scores are less affected by baseline fluorescence changes compared to gap scores, which is particularly advantageous for normalizing data under experimental conditions with varying background signals. This robustness of ratio scores in accounting for experimental variability ensures consistent performance, particularly in high-throughput or comparison settings. Statistical analysis confirmed significant differences between gap and ratio scores at each receptor concentration level (p < 0.01 in all comparisons). In particular, the performance at low receptor concentrations at 1 U demonstrates the sensitivity and potential applicability of the biosensor in scenarios requiring precise detection of ligand-receptor interactions, such as drug screening or therapeutic analysis.

[0113]

[0114] 3. Evaluation of the activity of modified EPO

[0115] To evaluate whether the biosensor could distinguish between natural EPO and structurally modified EPO, modified EPO was prepared by heating natural EPO at 95°C for 20 minutes. Evaluation using gap score and ratio score indices over 120 minutes showed that modified EPO exhibited a significantly smaller increase in both scores compared to natural EPO (Fig. 7). These observations demonstrate that the biosensor can effectively distinguish between active and inactive ligands. The difference in gap and ratio scores is attributed to structural changes in EPO during denaturation. Natural EPO maintains a distinct three-dimensional structure necessary for binding to EPOR and inducing receptor dimerization. Thermal denaturation destroys this structure, causing a loss of specific binding affinity and activating the receptor dimerization process. This finding demonstrates the sensitivity of the biosensor in detecting biologically important forms of EPO and its derivatives.

[0116] The ability of the biosensor to distinguish between the active and inactive forms of EPO holds significant implications for both research and therapeutic applications. For instance, in clinical settings, denatured or degraded EPO can lead to reduced efficacy or unwanted side effects. This biosensor can serve as a rapid and cost-effective tool for quality control in EPO production and storage processes. Furthermore, in basic research, this system enables the precise evaluation of EPO variants or derivatives designed for improved stability or activity. Additionally, Gap and Ratio scores provide complementary insights into ligand-receptor interactions. While the Gap score reflects the absolute difference in signal intensity between the test and control groups, the Ratio score reflects relative variation, normalizing variability based on experimental conditions. The consistently low Ratio scores observed in denatured EPO suggest that structural integrity is a critical determinant of receptor activation efficiency. These findings validate the use of BiFC-based biosensors for high-sensitivity analysis of ligand-receptor interactions and enhance their potential utility in ligand design, optimization, and quality control workflows.

[0117]

[0118] 4. Test of EPO interactions between spGFP domains

[0119] Although the biosensor showed different results depending on the presence or absence of EPO, it was unclear whether EPO directly dimerizes the receptor's ectodomain to induce the assembly of spGFP. To rule out the possibility that EPO directly interacted with the spGFP domain to induce assembly, GFP_11_sfCherry3C, which lacks the EPOR ectodomain, was cloned and purified (Fig. 8).

[0120] Unlike other biosensor proteins, GFP_11_sfCherry3C exhibited solubility due to the absence of a receptor ectodomain. For normalization, GFP_11_sfCherry3C was mixed with 1 U concentration of EPOR_GFP_1-10 using PAGE profiling, and an analysis was performed to measure the Gap score and Ratio score. Both scores showed a lower increase in the group with added GFP_11_sfCherry3C.

[0121] The gap score reflects the absolute difference in fluorescence intensity between the experimental and control groups. The low gap score observed in GFP_11_mCherry is likely due to the stronger baseline red fluorescence intensity of sfCherry3C, which can mask subtle changes in the green fluorescence signal. On the other hand, the ratio score normalizes changes in fluorescence intensity relative to the control condition, thereby reducing the impact of these baseline changes. Consequently, the ratio score provides a more robust indicator for comparing biosensor activity among various constructs. The consistently low gap and ratio scores observed in the GFP_11_sfCherry3C variant highlight the importance of the EPOR ectodomain in facilitating ligand-induced assembly. Without the ectodomain, the spatial orientation and proximity of the spGFP fragments alone were insufficient to induce efficient reassembly. This observation is consistent with previous studies indicating that the receptor ectodomain not only mediates ligand binding but also stabilizes structural changes necessary for downstream signaling. The differential performance of gap scores and ratio scores highlights their complementary roles in evaluating biosensor activity. Gap scores are useful for absolute comparisons in systems with minimal reference signal variability, whereas ratio scores are more reliable in scenarios involving constructs with heterogeneous fluorescence intensities or diverse reference fluorescence characteristics. Future iterations of the biosensor can benefit from design modifications aimed at optimizing the signal-to-noise ratio. For example, engineering mCherry variants with reduced fluorescence intensity or adjusting linker lengths between domains can further enhance signal detection and improve overall analytical performance (Fig. 9).

[0122]

[0123] 5. Cloning of other fluorescent proteins in the biosensor system of the present invention

[0124] The expression, purification, and refolding of EPOR-mCitrine and EPOR-mTFP1 were confirmed. The expression and purification of the synthesized genes were analyzed using 15% SDS-PAGE gel electrophoresis. After IPTG induction, two distinct bands corresponding to the estimated molecular weight (54 kDa) were observed. Since neither protein was present in the soluble fraction of the bacterial lysate, they were expressed as inclusion bodies. The pelleted fractions were washed with detergent and dissolved in 6 M urea-containing buffer as described in the methods. Purification was performed via HiTrap chelating nickel affinity chromatography, followed by the removal of most impurities via HiTrapQ ion exchange chromatography to obtain the purified fusion proteins. The fractions identified by SDS-PAGE results in FPLC were collected and dialyzed for refolding. Refolding was first confirmed using UV fluorescence. Both fusion proteins exhibited strong fluorescence (Figs. 10 and 11).

Claims

1. A first fusion protein in which a first erythropoietin receptor (EPOR) ectodomain and a first split fluorescent protein fragment are fused; and It comprises a second fusion protein in which a second erythropoietin receptor (EPOR) ectodomain and a second split fluorescent protein fragment are fused, and A biosensor system for monitoring the dimerization of an EPOR receptor induced by a ligand, wherein the first and second ectododomains are dimerized upon ligand binding.

2. A biosensor system according to claim 1, wherein the first and second split fluorescent protein fragments are assembled upon dimerization of the first and second EPOR ectododomains to emit signal fluorescence.

3. A biosensor system according to claim 1, wherein the second split fluorescent protein fragment is additionally connected to a reference fluorescent protein.

4. A biosensor system according to claim 1, wherein the split fluorescent protein fragment is a first split green fluorescent protein (split-enhanced GFP, spEGFP) and a second split green fluorescent protein (split-enhanced GFP, spEGFP); or mCitrine and mTFP1.

5. A biosensor system according to claim 1, wherein the first split fluorescent protein fragment is composed of the amino acid sequence of SEQ ID NO. 1, and the second split fluorescent protein fragment is composed of the amino acid sequence of SEQ ID NO. 2 or 3.

6. A biosensor system according to claim 1, wherein the first split fluorescent protein fragment is composed of the amino acid sequence of SEQ ID NO. 4 and the second split fluorescent protein fragment is composed of the amino acid sequence of SEQ ID NO.

5.

7. A biosensor system according to claim 1, wherein the system operates in vitro.

8. A method for detecting a target ligand using the biosensor system of Claim 1, wherein A step of mixing the first fusion protein and the second fusion protein with a sample that includes or is expected to include a target ligand; and A target ligand detection method comprising the step of measuring signal fluorescence according to the assembly of the split fluorescent protein fragment in the above mixture.

9. In claim 8, the detection method utilizes the biosensor system of claim 3, and A target ligand detection method comprising the step of measuring signal fluorescence according to the assembly of the split fluorescent protein fragment in the mixture and the reference fluorescence of the reference fluorescent protein.

10. A method for detecting a target ligand according to claim 8, wherein the target ligand is natural erythropoietin (EPO).

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