Gene cassette for biosensor expression, method for constructing same, and method for detecting target ligand using same

Abstract

The present invention relates to a gene cassette expressing a fusion protein, a method for constructing same, and a method for detecting a target ligand, using same. When a target ligand binds to a target receptor ectodomain to form a dimer, a fused split fluorescent protein fragment is assembled to generate a fluorescence signal, thereby enabling rapid and simple monitoring of the presence or absence of the target ligand.

Description

Gene cassette for biosensor expression, method for manufacturing the same, and method for detecting target ligand using the same

[0001] The present invention relates to a gene cassette, a biosensor, and a method for detecting a target ligand, and more specifically, to a gene cassette expressing a fusion protein, a method for manufacturing the same, and a method for detecting a target ligand using the same.

[0002]

[0003] The binding of receptor proteins to ligands plays a pivotal role in cellular signaling and various biological processes. In particular, cell membrane receptor proteins are responsible for the critical function of detecting signals from the external environment and transmitting them into the cell. When ligands bind to these receptors, they often undergo dimerization or multimerization, thereby activating signaling pathways. Therefore, technologies for accurately and efficiently monitoring the interaction between specific ligands and receptors, as well as the resulting receptor dimerization, are of paramount importance in life science research, drug development, and diagnostics.

[0004] Conventionally, various techniques have been developed for receptor dimerization and ligand detection. For example, radioligand binding assays using radioisotopes, enzyme-linked immunosorbent assays (ELISA), and Western blots using antibodies have been commonly used. However, these methods have several fundamental problems.

[0005] First, most conventional technologies require complex and multi-step processes. For example, ELISA requires multiple washing and incubation steps, and Western blot involves time-consuming steps such as protein extraction, electrophoresis, transfer, and antibody reaction. This delays detection time, requires operation by skilled technicians, and can increase variability in results.

[0006] Second, conventional analytical methods make it difficult to observe receptor dimerization in real time. Since the continuous measurement of the dynamic dimerization process occurring after ligand binding over time is limited, it is difficult to obtain important information such as reaction kinetics. This poses a significant limitation, particularly in understanding the mechanism of action of new drug candidates.

[0007] Third, some conventional technologies require expensive equipment or special reagents. For example, methods using Fluorescence Resonance Energy Transfer (FRET) require high-performance fluorescence microscopes or readers, which increases detection costs. In addition, methods using radioisotopes entail safety issues regarding handling and disposal, as well as the possibility of environmental contamination.

[0008] Fourth, conventional technology has room for improvement in terms of detection sensitivity and specificity. In particular, to directly monitor receptor dimerization in a cellular environment, high-sensitivity technology unaffected by the complex intracellular environment is required. Existing immunoassays may exhibit high background signals due to non-specific reactions, and quantitative analysis may be difficult.

[0009] Therefore, there is an urgent need for the development of new biosensor technology capable of simply, rapidly, and sensitively detecting the presence of a target ligand through the dimerization phenomenon of the target receptor, as well as gene cassettes to implement such technology.

[0010]

[0011] The present invention was carried out with the support of the following project.

[0012] [Project ID] 2460000771

[0013] [Assignment No.] KH141178

[0014] [Ministry Name] Ministry of Health and Welfare

[0015] [Name of Project Management (Specialized) Agency] Korea Health Industry Development Institute

[0016] [Research Project Name] Innovative Growth Skin Health Base Technology Development Project

[0017] [Project Title] Development of Novel Whitening Materials through Research on the Mechanism of Action of Erythropoietin on the Skin

[0018] [Name of Project Performing Organization] Daegu Gyeongbuk Institute of Science and Technology

[0019] [Research Period] 2024.01.01 ~ 2024.12.31

[0020]

[0021] The technical problem that the present invention aims to solve is to provide a gene cassette for biosensor expression.

[0022] In addition, the technical problem to be solved by the present invention is to provide a biosensor for monitoring the dimer formation of a target receptor by a target ligand and a method for detecting a target ligand using the same.

[0023]

[0024] 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.

[0025]

[0026] To achieve the above technical objectives, one embodiment of the present invention provides a gene cassette for biosensor expression comprising a first gene cassette expressing a first fusion protein and a second gene cassette expressing a second fusion protein, wherein the first gene cassette comprises a first gene sequence encoding a first target receptor ectodomain and a second gene sequence encoding a first split fluorescent protein fragment fused thereto, and the second fusion protein comprises a third gene sequence encoding a second target receptor ectodomain and a fourth gene sequence encoding a second split fluorescent protein fragment fused thereto, and wherein the first and second target receptor ectodomains comprise sequences that are identical or complementary to each other and become dimerized upon ligand binding.

[0027] In an embodiment of the present invention, the first and second target receptor ectodomains may be ectodomains of the erythropoietin receptor (EPOR), fibroblast growth factor receptor (FGFR), or platelet-derived growth factor receptor (PDGFR).

[0028] In an embodiment of the present invention, the first and third gene sequences may be configured to include restriction enzyme cleavage sites at both ends of the ectodomain gene sequence so as to allow the ectodomain gene sequence to be replaced with another sequence.

[0029] In an embodiment of the present invention, the first and third gene sequences may be any one of the nucleotide sequences of SEQ ID NOs 1 to 3.

[0030] In an embodiment of the present invention, the second and fourth gene sequences may be configured to include restriction enzyme cleavage sites at both ends of the split fluorescent protein fragment gene sequence, so as to enable the split fluorescent protein fragment gene sequence to be replaced with another sequence.

[0031] In an embodiment of the present invention, the fourth gene sequence may additionally have a fifth gene sequence encoding a reference fluorescent protein connected thereto.

[0032] In an embodiment of the present invention, the first and second split fluorescent protein fragments 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.

[0033] In an embodiment of the present invention, the second gene sequence may be a nucleotide sequence consisting of SEQ ID NO. 4, and the fourth gene sequence may be a nucleotide sequence consisting of SEQ ID NO. 5 or 6.

[0034] In an embodiment of the present invention, the second gene sequence may be a nucleotide sequence consisting of SEQ ID NO. 7, and the fourth gene sequence may be a nucleotide sequence consisting of SEQ ID NO. 8.

[0035] To achieve the above technical objective, another embodiment of the present invention provides a biosensor for monitoring the dimerization of a target receptor by a target ligand, manufactured according to the gene cassette.

[0036] In an embodiment of the present invention, the biosensor may be such that the first and second split fluorescent protein fragments are assembled upon dimerization of the first and second target receptor ectododomains to emit signal fluorescence.

[0037] In an embodiment of the present invention, the biosensor may be operated in vitro.

[0038] To achieve the above technical objective, another embodiment of the present invention provides a target ligand detection method using the biosensor, 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.

[0039] In an embodiment of the present invention, the detection method utilizes a biosensor manufactured according to the gene cassette, 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.

[0040]

[0041] The present invention relates to a gene cassette expressing a fusion protein, a method for manufacturing the same, and a method for detecting a target ligand using the same. Since the principle is utilized in which a fused split fluorescent protein fragment is assembled to generate a fluorescent signal when a target ligand binds to a target receptor ectodomain and they are dimerized, the presence of the target ligand can be monitored quickly and simply.

[0042] In addition, according to one embodiment of the present invention, by configuring the target receptor ectodomain and the split fluorescent protein fragment to be easily interchangeable using a restriction enzyme cleavage site, biosensors for various ligand-receptor pairs can be manufactured in a modular manner, thereby increasing applicability and versatility.

[0043] In addition, according to one embodiment of the present invention, by providing a gene cassette further comprising a reference fluorescent protein and a detection method using the same, the signal fluorescence can be normalized to the reference fluorescence, thereby minimizing the influence of non-specific background signals or dilution deviations between samples and improving the accuracy and reliability of quantitative analysis.

[0044]

[0045] 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.

[0046]

[0047] Figure 1 shows the design of a biosensor system using an spGFP assembly system.

[0048] Figure 2 briefly illustrates a strategy for cloning other receptors.

[0049] 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.

[0050] 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).

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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).

[0055] 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.

[0056] Figures 10 and 11 show the results of cloning another fluorescent protein in a biosensor system according to an embodiment of the present invention.

[0057] Figures 12 and 13 show the results of evaluating whether it can be generalized to other receptor-ligand systems.

[0058]

[0059] The present invention will be described in detail below.

[0060]

[0061] The present invention relates to a gene cassette for biosensor expression.

[0062] The present invention comprises a first gene cassette expressing a first fusion protein and a second gene cassette expressing a second fusion protein, wherein the first gene cassette comprises a first gene sequence encoding a first target receptor ectodomain and a second gene sequence encoding a first split fluorescent protein fragment fused thereto, and the second fusion protein comprises a third gene sequence encoding a second target receptor ectodomain and a fourth gene sequence encoding a second split fluorescent protein fragment fused thereto, and wherein the first and second target receptor ectodomains comprise sequences that are identical or complementary to each other and become dimerized upon ligand binding.

[0063] The gene cassette of the present invention may be a functional unit comprising a promoter, a coding sequence, a termination sequence, etc., necessary for gene expression. Such a gene cassette includes a first gene cassette expressing a first fusion protein and a second gene cassette expressing a second fusion protein. By using two separate cassettes, there may be an advantage of independently controlling the expression levels of the first and second fusion proteins or being able to use them flexibly in different host cell systems.

[0064] The first and second fusion proteins each comprise a first and second target receptor ectodomain and a first and second split fluorescent protein fragment. The ectodomain is a portion of the receptor exposed to the extracellular environment and can perform the function of binding to a ligand. The split fluorescent protein fragments do not exhibit fluorescence on their own, but 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 the ectodomain and induces dimerization, the split fluorescent protein fragments that come in close proximity are assembled to generate a fluorescent signal.

[0065] The first and second target receptor ectodomains described above include sequences that are identical or complementary to each other and become dimers 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. Such receptor ectodomains may be used without limitation as erythropoietin receptors (EPOR), fibroblast growth factor receptors (FGFR), platelet-derived growth factor receptors (PDGFR), or ectodomains of other tyrosine kinase receptor (RTK) families belonging to the same family (e.g., ErbB receptor, VEGF receptor, insulin receptor, etc.), or ectodomains of cytokine receptor families (e.g., interleukin receptor, growth hormone receptor, etc.). Preferably, the first and second target receptor ectodomens may be ectodomens of the erythropoietin receptor (EPOR), fibroblast growth factor receptor (FGFR), or platelet-derived growth factor receptor (PDGFR).

[0066] The first and third gene sequences above may be configured to include restriction enzyme cleavage sites at both ends of the ectodomain gene sequence, thereby allowing the ectodomain gene sequence to be replaced with another sequence. Restriction enzyme cleavage sites are technical configurations commonly used in molecular cloning and can perform the function of recognizing and cleaving specific nucleotide sequences. This has technical significance in that it enables the rapid fabrication of biosensors for various target ligands by easily replacing various ectodomains through the platformization of a gene cassette.

[0067] The above first and third gene sequences may specifically be any one of the nucleotide sequences of Sequence Nos. 1 to 3.

[0068] The second and fourth gene sequences may be configured to include restriction enzyme cleavage sites at both ends of the split fluorescent protein fragment gene sequence, thereby enabling the split fluorescent protein fragment gene sequence to be replaced with another sequence. Likewise, such a configuration allows for easy modification of the type of split fluorescent protein pair, which may be useful for optimizing sensor characteristics such as the signal-to-background ratio, fluorescence wavelength, and fluorescence intensity of the sensor.

[0069] The above-mentioned fourth gene sequence may be additionally linked with a fifth gene sequence encoding a reference fluorescent protein. More specifically, the third, fourth, and fifth gene sequences may be 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.

[0070]

[0071] The above-mentioned split fluorescent protein 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.

[0072] 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.

[0073] For a specific example, the second gene sequence may be a nucleotide sequence consisting of SEQ ID NO. 4, and the fourth gene sequence may be a nucleotide sequence consisting of SEQ ID NO. 5 or 6. Additionally, the second gene sequence may be a nucleotide sequence consisting of SEQ ID NO. 7, and the fourth gene sequence may be a nucleotide sequence consisting of SEQ ID NO. 8.

[0074] The present invention relates to a biosensor for monitoring the dimer formation of a target receptor by a target ligand, manufactured according to the gene cassette.

[0075] As for the gene cassette, it is as described above.

[0076] The above biosensor may be one in which first and second split fluorescent protein fragments are assembled upon the dimerization of first and second target receptor ectodomains to emit signal fluorescence. This may be the core operating principle of the sensor, which directly reports the presence of a ligand as the presence and intensity of a fluorescent signal. The assembly of the split fluorescent proteins can extremely sensitively reflect structural changes (i.e., dimerization) of the receptor ectodomains resulting from the binding of the ligand.

[0077] The above biosensor may operate in vitro. This means that the interaction and dimerization of the ligand and receptor can be observed without using cells, and it may have technical significance in that it eliminates the complexity of cell culture and processing and increases 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.

[0078] The present invention relates to a method for detecting a target ligand using the biosensor, 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.

[0079] Detailed explanations have been omitted for parts that overlap with the aforementioned content.

[0080] As a method for detecting a target ligand using the biosensor described above, 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, and the cost of protein preparation.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] When the above detection method uses a biosensor manufactured according to a gene cassette containing a reference fluorescent gene, the measuring step may include the 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.

[0085] 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.

[0086] 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.

[0087]

[0088] Hereinafter, in order to specifically explain the present invention, it will be described in detail with reference to examples.

[0089]

[0090] Materials and Methods

[0091] 1. Design of a Biosensor System Using an SpGFP Assembly System

[0092] In the case of bimolecular fluorescence complementation (BiFC) using the spEGFP system, spEGFP1-10 (SEQ No. 4) was linked to the EPOR ectodomain (SEQ No. 1) molecule, and spEGFP11 (SEQ No. 5) was linked to the EPOR ectodomain and sfCherry3C (spEGFP11-sfCherry3C, SEQ No. 6). The EPOR ectodomain and spEGFP are connected by a short loop containing the DNA cleavage site of EcoRI, which was designed to allow the ectodomain portion to be exchanged for 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).

[0093]

[0094] 2. Gene Synthesis

[0095] The plasmids used in this study and their corresponding maps are presented in Table 1 and Figure 1. Genes encoding EPOR-mCitrine (Sequence No. 13) or EPOR-mTFP1 (Sequence No. 15) 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).

[0096]

[0097] 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 / )

[0098] 3. Target gene cloning

[0099] 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.

[0100]

[0101] 4. Transformation, Culture, and Overexpression in E. coli Systems

[0102] 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.

[0103]

[0104] 5. Purification of target protein

[0105] 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.

[0106] 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.

[0107] Both soluble and insoluble proteins were finally dialyzed to 50 mM Tris-HCl (pH 8.0) and applied to the analysis system (Fig. 4).

[0108]

[0109] 6. Normalization of the molar ratio of sensor molecules

[0110] 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.

[0111] 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.

[0112]

[0113] 7. Detection of signal emission in biosensor systems

[0114] 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' minus '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).

[0115]

[0116] The sequence used in the present invention is as follows.

[0117]

[0118] result

[0119] 1. Biosensor Characteristics

[0120] 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.

[0121] 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.

[0122]

[0123] 2. Confirmation of biosensor activity

[0124] 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 the ligand-bound receptor complex 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.

[0125] 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.

[0126] 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.

[0127]

[0128] 3. Evaluation of the activity of modified EPO

[0129] 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.

[0130] 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.

[0131]

[0132] 4. Test of EPO interactions between spGFP domains

[0133] 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).

[0134] 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.

[0135] 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).

[0136]

[0137] 5. Cloning of other fluorescent proteins in the biosensor system of the present invention

[0138] 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).

[0139]

[0140] 6. Evaluate whether it can be generalized to other receptor-ligand systems.

[0141] To evaluate whether the biosensor platform could be generalized to other receptor-ligand systems, the EPOR ectodomain was replaced with the ectodomains of the fibroblast growth factor receptor (FGFR) and platelet-derived growth factor receptor (PDGFR) (Figs. 12 and 13). FGFR, a tyrosine kinase receptor, was selected because its structure is well-known and it is known to play a role in various biological processes such as cell proliferation and differentiation. PDGFR, another tyrosine kinase receptor, was also selected for the same reasons as FGFR. The cloning process involved inserting the FGFR ectodomain (SEQ No. 2) or the PDGF ectodomain (SEQ No. 3) into the spGFP biosensor system using the cloning tool of Infusion master mix (TAKARA).

Claims

1. A first gene cassette expressing a first fusion protein and a second gene cassette expressing a second fusion protein, and The first gene cassette comprises a first gene sequence encoding a first target receptor ectodomain and a second gene sequence encoding a first split fluorescent protein fragment fused thereto, and The second fusion protein comprises a third gene sequence encoding a second target receptor ectodomain and a fourth gene sequence encoding a second split fluorescent protein fragment fused thereto, and A gene cassette for biosensor expression, wherein the first and second target receptor ectododomains include sequences identical or complementary to each other and dimerize upon ligand binding.

2. The gene cassette of claim 1, wherein the first and second target receptor ectododomains are ectododomains of erythropoietin receptor (EPOR), fibroblast growth factor receptor (FGFR), or platelet-derived growth factor receptor (PDGFR).

3. A gene cassette according to claim 1, wherein the first and third gene sequences comprise restriction enzyme cleavage sites at both ends of the ectodomain gene sequence, configured to allow the ectodomain gene sequence to be replaced with another sequence.

4. A gene cassette according to claim 1, wherein the first and third gene sequences are nucleotide sequences of any one of SEQ ID NOs 1 to 3.

5. A gene cassette according to claim 1, wherein the second and fourth gene sequences comprise restriction enzyme cleavage sites at both ends of the split fluorescent protein fragment gene sequence, configured to allow the split fluorescent protein fragment gene sequence to be replaced with another sequence.

6. The gene cassette of claim 1, wherein the fourth gene sequence is additionally linked with a fifth gene sequence encoding a reference fluorescent protein.

7. In Claim 1, The first and second split fluorescent protein fragments are the first split green fluorescent protein (split-enhanced GFP, spEGFP) and the second split green fluorescent protein (split-enhanced GFP, spEGFP); or a gene cassette that is mCitrine and mTFP1.

8. A gene cassette according to claim 1, wherein the second gene sequence is a nucleotide sequence consisting of SEQ ID NO. 4, and the fourth gene sequence is a nucleotide sequence consisting of SEQ ID NO. 5 or 6.

9. A gene cassette according to Claim 1, wherein the second gene sequence is a nucleotide sequence consisting of SEQ ID NO. 7 and the fourth gene sequence is a nucleotide sequence consisting of SEQ ID NO.

8.

10. A biosensor for monitoring the dimer formation of a target receptor by a target ligand, manufactured according to the gene cassette of Claim 1.

11. The biosensor of claim 10, wherein the first and second split fluorescent protein fragments are assembled upon dimerization of the first and second target receptor ectododomains to emit signal fluorescence.

12. The biosensor of claim 10, wherein the biosensor operates in vitro.

13. A method for detecting a target ligand using the biosensor of claim 10, 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; 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.

14. In claim 10, the detection method uses a biosensor manufactured according to the gene cassette of claim 6, 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.

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