A novel protein specifically binding to small molecule HBC620 and inducing fluorescence enhancement and application thereof

Abstract

The present application relates to a kind of novel protein and its application specially combined with small molecule HBC620 and induced its fluorescence enhancement.The amino acid sequence of the protein is as shown in SEQ ID NO:1.The protein obtained in the present application can effectively reduce the non-specific binding with other structure similar small molecules due to the directional design of binding pocket around HBC620, thereby reducing the background fluorescence interference, improving detection accuracy and system stability.

Description

Technical Field

[0001] This invention belongs to the field of biosensing and molecular probe technology, specifically relating to a novel de novo protein that can specifically bind to the small molecule compound HBC620 and induce HBC620 to generate a fluorescent signal, and the use of this protein in constructing a fluorescence sensing system. Background Technology

[0002] Fluorescent probes are widely used in bioanalysis, medical diagnostics, environmental monitoring, and cell imaging due to their advantages such as high sensitivity, fast detection speed, ease of operation, and real-time imaging capabilities (Lakowicz JR., Principles of Fluorescence Spectroscopy, Springer, 2006). Among them, "activation-type" fluorescent systems formed by the binding of small-molecule fluorescent compounds with specific recognition elements can effectively reduce background signals and improve the detection signal-to-noise ratio, and have received widespread attention in recent years (Chan J. et al., Reaction-based small-molecule fluorescent probes for chemoselective bioimaging, Nat Chem, 2012, 4: 973-984).

[0003] While traditional fluorescent proteins (such as green fluorescent protein and its derivatives) can achieve bioimaging, they typically suffer from drawbacks such as large molecular weight, complex maturation processes, limited spectral modulation, and sensitivity to the cellular environment. In contrast, fluorescence enhancement systems constructed using small-molecule fluorescent compounds and specifically binding proteins combine the advantages of high brightness, tunable spectra, and strong protein recognition specificity of small-molecule fluorescent dyes, and have become an important direction for the development of novel fluorescence sensing technologies (Lavis L.D. et al., Bright ideas for chemical biology, ACS Chem Biol, 2008, 3: 142-155).

[0004] Currently, some small-molecule fluorescence enhancement systems based on natural or engineered proteins have been reported. For example, these systems restrict the conformational freedom of fluorescent molecules through protein binding, thereby reducing nonradiative transitions and enhancing fluorescence signals (Paige J.S. et al., RNA mimics of green fluorescent protein, Science, 2011, 333(6042): 642-646). However, most of the proteins in existing technologies are derived from modified natural proteins, and their binding pocket structures are limited by the natural backbone. This often results in insufficient adaptability to target small molecules, limited fluorescence enhancement efficiency, low specificity, and insufficient stability. Furthermore, for specific novel fluorescent small molecules, there is a lack of protein systems that can bind with high specificity and effectively induce fluorescence enhancement.

[0005] HBC620 (CAS: 2530162-07-1, ccd code: J93, λ ex 577 nm, λ em HBC620 (620 nm) is a class of small molecule compounds with potential value in bioimaging and sensing applications. While its fluorescence is weak in its free state, it can exhibit significant fluorescence enhancement in specific microenvironments. Therefore, developing novel proteins that can specifically bind to HBC620 and induce strong fluorescence signals is of great significance for constructing novel fluorescence sensing systems with high sensitivity and low background.

[0006] In recent years, with the development of AI-assisted protein design and de novo protein design technologies, researchers have been able to directly design artificial proteins with specific structures and functions without relying on natural protein templates. This provides a new technical pathway for constructing novel small-molecule binding proteins (Jumper J. et al., Highly accurate protein structure prediction with AlphaFold, Nature, 2021, 596: 583-589; Daupras J. et al., Robust deep learning-based protein sequence design using ProteinMPNN, Science, 2022, 378: 49-6). Currently, proteins that can specifically bind to HBC620 and significantly enhance its fluorescence signal, as well as their related application systems, are all based on multiple rounds of experimental directed evolution (Jingyi Z. et al., Denovo design of transmembrane fluorescence-activating proteins, Nature 2025, 640: 249-257). A de novo-designed specific HBC620-binding protein is still lacking. Summary of the Invention

[0007] To address the problems of existing small molecule fluorescence enhancement systems, such as reliance on natural protein modification, long development cycles, insufficient adaptability to specific small molecules, and the need for multiple rounds of experimental directed evolution, this invention aims to provide a novel de novo-designed protein that can specifically bind to the small molecule HBC620 and induce significant fluorescence enhancement, as well as its applications.

[0008] Specifically, the present invention provides the following technical solutions.

[0009] On the one hand, the present invention provides a protein that specifically binds to the small molecule HBC620, the amino acid sequence of which is shown in SEQ ID NO:1.

[0010] On the other hand, the present invention provides polynucleotides that encode proteins as described above.

[0011] On the other hand, the present invention provides an expression vector comprising the polynucleotides described above.

[0012] On the other hand, the present invention provides a host cell containing the expression vector as described above.

[0013] In some embodiments, the host cell is selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Bacillus, and insect cells. Preferably, the host cell is Escherichia coli.

[0014] On the other hand, the present invention provides a sensor comprising the protein as described above.

[0015] On the other hand, the present invention provides a kit containing the protein as described above.

[0016] On the other hand, the present invention provides a fusion protein comprising the protein as described above and a target polypeptide fused with the protein.

[0017] On the other hand, the present invention provides a fluorescence sensing system comprising:

[0018] (a) the protein as described above, or the fusion protein as described above; and

[0019] (b) Small molecule compound HBC620.

[0020] On the other hand, the present invention provides a method for detecting target molecules, the method comprising:

[0021] (a) The fusion protein described above is brought into contact with the sample to be tested, wherein the target polypeptide in the fusion protein is a binding domain capable of specifically recognizing the target molecule;

[0022] (b) Add the small molecule compound HBC620;

[0023] (c) Detect fluorescence signal.

[0024] On the other hand, the present invention provides the application of the proteins, polynucleotides, expression vectors or host cells as described above in imaging and sensing.

[0025] On the other hand, the present invention provides an imaging method that uses the protein or fusion protein described above.

[0026] On the other hand, the present invention provides the application of combinations of proteins, polynucleotides, expression vectors, host cells or fusion proteins as described above with HBC620 in imaging and sensing.

[0027] On the other hand, the present invention provides an imaging method that uses a combination of the protein or fusion protein and HBC620 as described above.

[0028] Beneficial effects

[0029] Compared with existing small molecule fluorescence enhancement systems based on natural protein modification or multiple rounds of directed evolution, the artificially designed HBC620 binding protein provided by this invention has the following advantages and positive effects:

[0030] (1) The protein described in this invention does not rely on a natural protein template, but is constructed artificially from scratch to form a specific small molecule binding pocket. Therefore, it is possible to target the molecular structure and physicochemical characteristics of HBC620, thereby improving the spatial matching and interaction strength between the binding site and HBC620. Compared with the limitations of natural protein modification methods due to the original backbone, this invention has higher structural designability and functional controllability.

[0031] (2) This invention constructs a small molecule binding cavity encapsulating HBC620, restricting the conformational freedom of the HBC620 molecule and altering its local microenvironment, thereby reducing nonradiative transitions and increasing fluorescence quantum yield. Experimental results show that after HBC620 binds to the protein of this invention, its fluorescence signal is significantly enhanced compared to the free state, forming a fluorescence response system with low background and high signal-to-noise ratio.

[0032] (3) The protein obtained by this invention has high binding specificity. Because the binding pocket is oriented around HBC620, it can effectively reduce non-specific binding with other structurally similar small molecules, thereby reducing background fluorescence interference and improving detection accuracy and system stability.

[0033] (4) The present invention uses an artificial intelligence-assisted protein design and structure prediction method to screen candidate proteins. Compared with the traditional method that relies on random mutation and multiple rounds of directed evolution, it can reduce the workload of experimental screening, shorten the development cycle, and improve the efficiency of obtaining target proteins.

[0034] (5) The protein obtained by this invention has good physicochemical properties, including good solubility, thermal stability, and expression stability. The secondary structure stability of the protein was determined by circular dichroism (CD), and the results showed that the protein could maintain stable folding under high temperature conditions, with a thermal denaturation temperature (Tm) of approximately 85℃. High thermal stability is beneficial for maintaining structural and functional stability of the protein under different storage, transportation, and application conditions, improving its reliability in fluorescence detection, biosensing, and in vitro analysis systems.

[0035] (6) The HBC620 fluorescence enhancement system constructed in this invention has long wavelength excitation and emission characteristics (λex about 535 nm, λem about 595 nm), which is beneficial to reduce the background of autofluorescence in biological samples and improve the signal-to-noise ratio in the imaging and detection process. Therefore, it has application value in the fields of biological imaging, fluorescence detection and molecular sensing.

[0036] (7) The protein obtained by the present invention can be further engineered as a modular small molecule recognition element and can be combined with other protein modules, target molecules or biosensing systems to construct novel fluorescent labeling systems, live cell sensing systems and analysis and detection platforms. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the HBC620 small molecule.

[0038] Figure 2 The predicted structure of the HBC620-binding protein complex with a small molecule is shown.

[0039] Figure 3 The results of HBC620 binding protein absorption and emission spectra measurements are shown.

[0040] Figure 4 The results of HBC620 binding protein thermal stability assays are shown.

[0041] Figure 5 The HBC620 binding protein affinity fitting curve is shown.

[0042] Figure 6 The changes in fluorescence signal are shown when HBC620 is present alone and after the addition of HBC620-specific binding protein. Detailed Implementation

[0043] The embodiments of this application will be clearly and completely described below. The technical solutions in the embodiments described below are exemplary and only possible technical solutions of this application, not all possible technical solutions. Those skilled in the art can combine the embodiments of this application to obtain other embodiments without creative effort, and these embodiments are also within the protection scope of this application.

[0044] In this invention, the term "thermal denaturation temperature" refers to the critical temperature at which the spatial conformation (secondary, tertiary, and quaternary structures) of a protein undergoes irreversible damage, resulting in the loss of its biological activity. The thermal denaturation temperature of ordinary proteins is typically between 40-60°C, while that of heat-resistant proteins is greater than 70°C. The protein of this invention has a thermal denaturation temperature of approximately 85°C, classifying it as an extremely heat-resistant protein that is not easily inactivated under normal storage or handling conditions.

[0045] In this invention, the term "HBC620" refers to a non-fluorescent HBC analog that can emit strong fluorescence when bound to a specific substance.

[0046] In this invention, materials with "longer wavelength excitation and emission characteristics" typically refer to systems with an excitation wavelength ≥ 400 nm (visible light) and an emission wavelength entering the red / near-infrared region. These materials effectively avoid interference from the autofluorescence of biological tissues and penetrate deeper, offering significant advantages in the field of bioimaging.

[0047] In this invention, far-ultraviolet circular dichroism (CD) spectroscopy is the core technology for characterizing protein secondary structures (α-helices, β-sheets). The wavelength range is 190-250 nm, where peptide bonds (amide chromophores) generate CD signals. α-helices exhibit double negative peaks at 208 nm and 222 nm, β-sheets exhibit a single negative peak at 215-218 nm, and random coils exhibit a negative peak near 198 nm. Far-ultraviolet circular dichroism (CD) spectroscopy can be used to rapidly determine whether a protein is folded, the proportion of its secondary structure, and its thermo / chemical stability.

[0048] In this invention, "goodness of fit" is an important statistical indicator used to measure how well a regression model fits the observed data, primarily measured by the coefficient of determination R. 2 To represent. R 2 The closer to 1, the better the model fit.

[0049] In this invention, "LigandMPNN" is a deep learning model for protein design, based on an extension of the ProteinMPNN architecture, and optimized specifically for ligand-binding protein design. It was released in 2023 by David Baker's team (Protein Design Institute, University of Washington, IPD) and aims to design protein sequences that interact with small molecules, peptides, or other ligands.

[0050] In this invention, "Alphafold3" is an artificial intelligence model for predicting biomolecular structures, jointly developed by Google DeepMind and Isomorphic Labs, and first published in the journal Science on May 8, 2024. This model is an improvement on the AlphaFold2 architecture, using a diffusion module to directly predict atomic coordinates and introducing a Pairformer module to reduce dependence on multiple sequence alignment (MSA). It can predict the three-dimensional structures and interactions of biomolecules such as proteins, DNA, RNA, and ligands.

[0051] The purpose of this invention is to construct a protein with a stable spatial structure and a specific binding pocket through artificial protein design technology, enabling it to efficiently recognize and bind to HBC620. This is achieved by restricting the conformational freedom of the HBC620 molecule, suppressing its intramolecular torsion rotation and vibration, improving molecular planarity, and embedding it in a relatively hydrophobic and rigid binding microenvironment. This reduces water quenching and non-radiative energy dissipation, thereby significantly improving the fluorescence intensity of HBC620 and forming a low-background, high-signal-to-noise ratio fluorescence sensing system. The low background is reflected in the extremely weak fluorescence of HBC620 in its free state, with significant fluorescence enhancement only occurring after binding to the target protein. The high signal-to-noise ratio is reflected in the significant difference in fluorescence signal before and after binding, effectively reducing background interference and improving detection sensitivity. Furthermore, this system exhibits good thermal stability, solubility, and structural stability. This invention also shows promise for applications in fluorescence detection, biosensing, molecular imaging, target molecule recognition, and related analytical detection fields, providing a new technical solution and application foundation for constructing novel small-molecule fluorescent probe systems.

[0052] Specifically, this invention provides a novel protein capable of specifically binding to the small molecule HBC620 and inducing its fluorescence enhancement, as well as its applications. The protein is a de novo designed protein, independent of natural protein templates. A protein backbone with a specific spatial structure and small molecule binding pocket is constructed using computer-aided protein design methods, and sequence optimization yields a protein with good expressibility, stability, and HBC620 binding ability.

[0053] The protein obtained by this invention contains a combination of amino acid residues that can form a binding cavity for HBC620. This binding cavity can interact with HBC620 through hydrophobic interactions, hydrogen bonding, π-π interactions, and electrostatic interactions, thereby restricting the conformational freedom of the HBC620 molecule and altering its local microenvironment, reducing nonradiative transitions, and inducing HBC620 to generate an enhanced fluorescence signal.

[0054] The protein obtained in this invention possesses stable secondary and tertiary structures. Its overall structure is a four-helix bundle of monomeric protein, with four α-helices connected by loop regions to form a tightly packed hydrophobic core, thus maintaining the overall stability of the protein. This hydrophobic core also serves as an ideal binding site for small molecules. Structural analysis results show that the protein's primary secondary structure is composed of α-helices, without any significant disordered regions. Structural prediction results indicate that the protein exists stably in monomeric form, with the HBC620 binding site located within the internal hydrophobic cavity formed by the four helices. Enhancement of HBC620 fluorescence properties is achieved through local microenvironment regulation.

[0055] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0056] Example 1: Design and Optimization of HBC620-Specific Binding Protein

[0057] The proteins of this invention are designed and optimized using artificial intelligence-assisted protein design methods, structure prediction methods, and sequence screening methods. Specifically, they include:

[0058] 1. Construct protein backbone structures with predetermined small molecule binding pockets using the independently developed protein backbone structure generation software SCUBA-T;

[0059] 2. The amino acid sequence of the designed protein backbone and small molecule structure was designed using the sequence reverse folding model LigandMPNN;

[0060] 3. The amino acid sequence and the complex structure of the small molecule HBC620 were predicted using the Alphafold3 structural prediction model. Based on the indicators predicted by Alphafold3, 37 amino acid sequences that met the indicators were obtained (screening indicators: Protein scRMSD < 1.5 Å, Ligand scRMSD < 5 Å, ipTM > 0.8, min ipAE < 1.5).

[0061] 4. The candidate protein was expressed, purified, and validated using fluorescence enhancement experiments;

[0062] 5. Obtain the target protein that can significantly enhance the HBC620 fluorescence signal and has high affinity, good stability and specificity, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0063] Example 2: Expression and purification of HBC620-specific binding protein

[0064] 1. The gene encoding the target protein (SEQ ID NO:2) was cloned into the pET-29b(+) expression vector (Beijing Huayueyang Biotechnology) and transformed into E. coli BL21(DE3) competent cells.

[0065] 2. Select positive single clones and inoculate them into LB medium containing antibiotics, and culture at 37°C with shaking until the OD600 is approximately 0.6-0.8.

[0066] 3. Add 0.5 mM IPTG to induce expression overnight at 16-20℃.

[0067] 4. Collect the bacterial cells by centrifugation and resuspend them in lysis buffer (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0).

[0068] 5. Cells are disrupted by sonication, followed by high-speed centrifugation to remove cell debris, and the supernatant is collected.

[0069] 6. Load the supernatant into a pre-equilibrated Ni-NTA affinity chromatography column and capture the target protein by utilizing the specific binding of the His tag to the Ni-NTA resin.

[0070] 7. Use washing buffer (20 mM Tris-HCl, 300 mM NaCl, 40 mM imidazole, pH 8.0) to remove non-specifically bound proteins.

[0071] 8. Elute the target protein using elution buffer (20 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole, pH 8.0).

[0072] 9. Collect the elution fraction and determine the purity of the target protein by SDS-PAGE. If necessary, further purification is performed using molecular sieve chromatography, and the sample is then transferred to a buffer suitable for subsequent fluorescence detection, which contains 300 mM NaCl, 20 mM Tris buffer, and pH 7.8.

[0073] Example 3: Determination of the thermal stability of HBC620-specifically binding proteins

[0074] The secondary structure and thermal stability of HBC620-specific binding proteins were detected by circular dichroism (CD).

[0075] The protein purified in Example 2 was replaced with 10 mM PBS buffer, pH 7.4. The protein concentration was adjusted to approximately 0.3 mg / mL and added to a quartz cuvette with a path length of 1 mm.

[0076] First, far-ultraviolet circular dichroism spectra were acquired at 25℃, with a scanning range of 190-260 nm. The results showed that the protein had obvious negative peaks near 208 nm and 222 nm, indicating that its main secondary structure is an α-helix, consistent with the structural characteristics of a four-strand α-helix protein.

[0077] Subsequently, the ellipticity signal at 222 nm was used as a monitoring indicator for temperature scanning. The test temperature range was 25-95 ℃, the heating rate was 1 ℃ / min, and the CD signal changes at 222 nm were continuously recorded. The results showed that the protein α-helix signal gradually weakened with increasing temperature, but a significant negative peak signal at 222 nm was still retained above 90 ℃, indicating that the protein has high thermal stability. According to the fitting of the thermal denaturation curve, its apparent melting temperature Tm is approximately 85 ℃, as shown in the figure. Figure 4 As shown.

[0078] Example 4: Fluorescence response assay of HBC620-specific binding protein

[0079] 1. Prepare a purified HBC620-specific binding protein solution to a final protein concentration of 10 μM.

[0080] 2. Prepare a small molecule working solution of HBC620 (GLPBIO, catalog number GC60186) and add it to the protein solution to make the final concentration of HBC620 10 μM.

[0081] 3. A control group was set up simultaneously: a small molecule solution containing only 10 μM HBC620, without the addition of protein.

[0082] 4. Add each group of samples to a black 96-well microplate, with a total volume of 100 μL per well and 3 parallel wells per group.

[0083] 5. Incubate the sample at room temperature in the dark for 30 min to allow the protein to fully bind with HBC620.

[0084] 6. Use an ELISA reader to detect the fluorescence signal, with the excitation wavelength set to 535 nm and the emission wavelength set to 595 nm.

[0085] 7. Record the fluorescence intensity of each group at 595 nm, and use the small molecule solution containing only HBC620 as the baseline control to calculate the fluorescence enhancement factor.

[0086] The results are as follows Figure 6 As shown, HBC620 exhibits a weak fluorescence signal when present alone, but the fluorescence signal at 595 nm is significantly enhanced after the addition of the HBC620-specific binding protein. With both the HBC620-specific binding protein and HBC620 at a final concentration of 10 μM, the fluorescence intensity of the protein-HBC620 complex is approximately 1400 times that of the HBC620 solution alone, indicating that the designed protein can bind to HBC620 and induce its fluorescence enhancement.

[0087] Example 5: Affinity determination of HBC620-specific binding proteins

[0088] 1. Prepare HBC620 working solution to a final concentration of 0.5 μM.

[0089] 2. The purified HBC620-specific binding protein was serially diluted using detection buffer (300 mM NaCl, 20 mM Tris buffer, pH 7.8) to final concentrations of 0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 32, 64, 128, and 256 μM.

[0090] 3. Mix equal volumes of HBC620 working solution with protein solutions of different concentrations and add the mixture to a black 96-well microplate. The total volume for each well is 100 μL, and three parallel wells are set for each concentration.

[0091] 4. Incubate the mixture at room temperature in the dark for 30 min to allow HBC620 to fully bind to the protein.

[0092] 5. Use an ELISA reader to detect the fluorescence signal, with the excitation wavelength set to 535 nm and the emission wavelength set to 595 nm.

[0093] 6. Using the untreated protein group as a background control, record the fluorescence intensity corresponding to each protein concentration and subtract the background signal.

[0094] 7. Using protein concentration as the x-axis and fluorescence intensity after background subtraction as the y-axis, a single-point binding model was used for nonlinear fitting.

[0095] The fitting results are as follows Figure 5 As shown, the apparent dissociation constant of the HBC620-specific binding protein with HBC620 is K. d =50.6 nM, goodness of fit R 2 > 0.99.

[0096] Example 6: Fluorescence spectroscopy determination of HBC620-specific binding protein

[0097] 1. Prepare a purified HBC620-specific binding protein solution to a final protein concentration of 10 μM.

[0098] 2. Prepare a small molecule working solution of HBC620 and add it to the protein solution to make the final concentration of HBC620 10 μM.

[0099] 3. Add each group of samples to a black 96-well microplate, with a total volume of 100 μL per well and 3 parallel wells per group.

[0100] 4. Incubate the sample at room temperature in the dark for 10-30 minutes to allow the protein to fully bind with HBC620.

[0101] 5. Use an ELISA reader to detect the fluorescence signal, set the excitation wavelength to 480nm, and measure the fluorescence intensity in the range of 560-700nm.

[0102] 6. Use an ELISA reader to detect the fluorescence signal. Set the excitation wavelength to the range of 400-570nm and measure the fluorescence intensity at 660nm.

[0103] The results are as follows Figure 3 As shown, the fluorescence enhancement system of the present invention has long wavelength excitation and emission characteristics, λex about 535 nm and λem about 595 nm.

[0104] sequence

[0105] SEQ ID NO:1 Amino acid sequence of HBC620 binding protein

[0106] SAAEERLALIEEVKKLVAEFQKLAGKAQAAFAAGRYDEVLAGAEKYLKEVEKLLEKATDEKLKAILEKIKAGLEQVIEAARLGAEGRQAEAAALLAQAQAQLGEVQKELEEYEKEFLASLW

[0107] SEQ ID NO:2 nucleotide sequence of HBC620 binding protein after adding histidine tag

[0108] ATGAGCGCAGCAGAAGAACGTCTGGCACTGATTGAAGAAGTTAAAAAAACTGGTGGCAGAGTTTCAGAAACTGGCCGGTAAAGCCCAGGCAGCATTTGCAGCCGGCCGTTATGATGAAGTTCTGGCCGGCGCAGAAAAATATCTGAAAGAAGTGGAAAAGCTGCTGGAAAAAGCAACCGATGAAAAACTGAAAGCC ATTCTGGAAAAGATCAAGGCAGGTCTGGAACAGGTTATTGAAGCAGCCCGTCTGGGTGCAGAAGGCAGACAAGCAGAAGCCGCAGCACTGCTGGCACAGGCACAAGCACAGCTGGGTGAAGTGCAGAAAGAACTGGAAGAATATGAAAAGGAGTTCCTGGCCAGCCTGTGGCTCGAGCACCACCACCACCACCAC

[0109] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A protein that specifically binds to the small molecule HBC620, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO:

1.

2. A polynucleotide, characterized in that, The polynucleotide encodes the protein according to claim 1.

3. An expression carrier, characterized in that, The expression vector comprises the polynucleotide according to claim 2.

4. A host cell, characterized in that, The host cell comprises the expression vector according to claim 3. Preferably, the host cell is selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Bacillus subtilis, and insect cells. Preferably, the host cell is Escherichia coli.

5. A reagent kit, characterized in that, The kit contains the protein according to claim 1.

6. A fusion protein, characterized in that, The fusion protein comprises the protein of claim 1 and the target polypeptide fused with the protein.

7. A fluorescence sensing system, characterized in that, The fluorescence sensing system comprises: (a) the protein of claim 1, or the fusion protein of claim 6; and (b) Small molecule compound HBC620.

8. An imaging method, characterized in that, The method is performed using the protein according to claim 1 or the fusion protein according to claim 6.

9. A method for detecting target molecules, characterized in that, The method includes: (a) Contact the fusion protein of claim 6 with the sample to be tested, wherein the target polypeptide in the fusion protein is a binding domain capable of specifically recognizing the target molecule; (b) Add the small molecule compound HBC620; (c) Detect fluorescence signal.

10. The application of the protein according to claim 1, the polynucleotide according to claim 2, the expression vector according to claim 3, the host cell according to claim 4, or the fusion protein according to claim 6 in imaging.

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