Enhanced green fluorescent protein mutant as well as preparation method and application thereof

Abstract

The invention discloses an enhanced green fluorescent protein mutant, and the amino acid sequence of the enhanced green fluorescent protein mutant has the following mutation sites: S30R, Y39N, N105T, I171V, A206V and L220F compared with the amino acid sequence of green fluorescent protein roGFP1 or roGFP2. The invention discloses a fluorescent probe. Raw materials for preparation comprise an enhanced green fluorescent protein mutant. The invention also discloses a preparation method of the enhanced green fluorescent protein mutant and application of the enhanced green fluorescent protein mutant in an oxidation sensor. Compared with green fluorescent protein, the enhanced green fluorescent protein mutant disclosed by the invention has the advantages that the fluorescence intensity is enhanced, the response range is expanded, and the enhanced green fluorescent protein mutant can be used as a strong and high-sensitivity fluorescent indicator for monitoring the redox potential in cells.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an enhanced green fluorescent protein mutant, its preparation method, and its application. Background Technology

[0002] Intracellular redox balance, determined by the relative levels of oxidants and reductants, plays a crucial role in regulating a wide range of cellular processes. Redox balance is tightly regulated to support a broad spectrum of fundamental cellular functions, including cell signaling, mitosis, immune responses, cell death, and metabolism. This complex control system maintains an appropriate balance (redox state) between oxidants and reductants in different subcellular compartments, which is essential for their specific functions. For example, the cytoplasm and mitochondria exhibit a relatively reducing environment characterized by a high reduced glutathione / oxidized glutathione (GSH / GSSG) ratio. This protects cysteine ​​residues in proteins from oxidative damage and supports reductive biochemical reactions. Conversely, the endoplasmic reticulum (ER) maintains a more oxidative redox potential, which is crucial for the proper formation of disulfide bonds during secretion and membrane protein folding. This separation underscores the importance of accurately measuring the redox potential difference between organelles to understand their unique physiological and pathological roles.

[0003] Genetically encoded redox-sensitive green fluorescent proteins (roGFPs) offer a robust, non-invasive approach for spatially and temporally resolving intracellular redox states. Their function is based on a conformational change caused by the formation (or reduction) of a disulfide bond between two engineered cysteine ​​residues (C147 and C204) located on adjacent β-chains of the GFP β-barrel structure. This dynamic change in the disulfide bond alters the protonation state of the pigment, resulting in a measurable shift in its excitation spectrum. By targeting roGFPs to specific organelles, redox potentials can be quantified based on excitation ratios. However, detecting redox potential dynamics in subcellular organelles and in vivo remains a significant challenge, primarily due to the low fluorescence intensity and limited dynamic range of currently available roGFP sensors. The relatively low fluorescence intensity and weak response limit their application in subcellular organelles such as the Golgi apparatus and in live animals. Summary of the Invention

[0004] The purpose of this invention is to provide an enhanced green fluorescent protein mutant to improve its fluorescence intensity and expand its response range.

[0005] To achieve the above objectives, the present invention discloses an enhanced green fluorescent protein mutant, whose amino acid sequence has the following mutation sites compared with the amino acid sequence of green fluorescent protein roGFP1 or roGFP2: S30R, Y39N, N105T, I171V, A206V, L220F.

[0006] Furthermore, the amino acid sequence of the enhanced green fluorescent protein mutant is shown in SEQ ID No:1 or SEQ ID NO:2.

[0007] The present invention also discloses a fluorescent probe, wherein the raw materials for preparing the fluorescent probe include the above-mentioned enhanced green fluorescent protein mutant.

[0008] The method for preparing the enhanced green fluorescent protein mutant of the present invention includes the following steps: Mutations were introduced at the mutation sites in green fluorescent protein roGFP1 or roGFP2: serine at position 30 was mutated to arginine, tyrosine at position 39 to asparagine, asparagine at position 105 to threonine, isoleucine at position 171 to valine, alanine at position 206 to valine, and leucine at position 220 to phenylalanine. All mutations were constructed using Prime Star DNA polymerase on the pRSETb vector via reverse PCR.

[0009] Furthermore, the constructed plasmid was expressed in Escherichia coli and induced to form a recombinant protein via isopropyl-β-thiogalactoside.

[0010] The present invention also discloses the use of enhanced green fluorescent protein mutants in oxidation sensors.

[0011] Furthermore, the oxidation sensor has any of the following uses: a. monitoring redox balance dynamics, b. real-time redox potential calibration, c. in-situ imaging, d. visualizing the redox potential of cells.

[0012] Based on the above solution, the present invention has the following beneficial effects: The enhanced green fluorescent protein mutant of the present invention has enhanced fluorescence intensity and expanded response range compared with green fluorescent protein. It can be used as a powerful and highly sensitive fluorescent indicator to monitor intracellular redox potential and study the redox regulation of thiol-disulfide in living cells and intact organisms.

[0013] The enhanced green fluorescent protein mutant (eroGFP1.2) mutated on roGFP1 exhibited fluorescence intensity more than five times higher than wild-type roGFP1 in E. coli and HeLa cells, with a 50% increase in dynamic range, and calibrated the redox potentials of different subcellular compartments within HeLa cells. Furthermore, the practicality of this invention in vivo was demonstrated by monitoring the reactive oxygen species (ROS) burst at the wound edge after caudal fin amputation in juvenile zebrafish. Attached Figure Description

[0014] Figure 1A This is a schematic diagram of the mutation sites in the enhanced green fluorescent protein mutant eroGFP1.2.

[0015] Figure 1B Modeling the 3D structure of eroGFP1.2.

[0016] Figure 1C This is a schematic diagram of the mutation sites in the enhanced green fluorescent protein mutant eroGFP2.2.

[0017] Figure 2 B represents the baseline brightness of the constructed mutant expressed in *E. coli*. Fluorescence in both excitation channels of the roGFP1 sensor was normalized to 1.0.

[0018] Figure 2 C represents the maximum dynamic change, calculated by the ratio of the oxidation sensor to the reduction sensor. This was achieved by adding 100 μM difluoride and 5 mM dithiothreitol (DTT) to the bacterial suspension and monitoring the changes using a fluorescent plate reader.

[0019] Figure 2 D shows representative fluorescence images of bacterial colonies of roGFP1 and its enhanced variants on LB plates. All indicators were transfected into *E. coli* and incubated overnight at 37°C. Images were taken 20 hours post-transfection using an In-Vivo multispectral system FX with two excitation filters, 400 / 10 (left) and 480 / 10 (right), and an emission filter at 530 nm.

[0020] Figure 2 E represents the fluorescence of roGFP2, eroGFP2.1, and eroGFP2.2 expressed in bacteria. Detection conditions are the same as in 2B.

[0021] Figure 3 B represents the baseline fluorescence of the *E. coli* mutant. The fluorescence of the two excitation channels of the Roggen FP1 sensor was normalized to 1.0.

[0022] Figure 3C represents the maximum change in the conversion of roGFP1 to 100 μM diamide and 5 mM dithiothreitol (DTT) normalized to roGFP1.

[0023] Figure 3 D represents the fluorescence of roGFP1 sensors with different superfolder mutations expressed in bacteria. (R: S30R; N: Y39N; T: N105T; V: I171V; F: Y145F; V: A206V).

[0024] Figure 3 E represents the fluorescent traces of roGFP1 and eroGFP1.2 in bacteria reacting with 0.1 mM diamide or 5 mM DTT.

[0025] Figure 3 F-3G represents the baseline fluorescence (F) and maximum variation (G) of roGFP2 and eroGFP2.0. Both are normalized to roGFP2.

[0026] Figure 4 A shows the fluorescence spectra of eroGFP1.2 after complete oxidation and reduction in the presence of 0.1 mM diamino and 5 mM DTT.

[0027] Figure 4 B represents the dynamic excitation ratio of 400 / 480, measured in vitro using a fluorescent plate reader. F 400 / F 480 The reduced roGFPs were normalized to 1. Unless otherwise specified, the following data are treated in the same manner. Error bars represent standard errors.

[0028] Figure 4 C determines the redox titration by plotting the relationship between the fraction of oxidized proteins and the equivalent redox potential. The midpoint redox potential of roGFPs is calculated by fitting data.

[0029] Figure 4 D represents the fluorescence ratio of fully oxidized (black circle) and fully reduced (red circle) eroGFP1.2 at different pH values. The buffer contains 100 mM MES (pH 5.5-6.5) or HEPES (pH 7.0-8.0) or TRICINE (pH 8.5) and 100 mM sodium chloride.

[0030] Figure 4 E represents the residual dual-channel fluorescence of the roGFP sensor after heat treatment at 90 °C for 30 minutes.

[0031] Figure 5A represents the dynamic changes of purified roGFP1, eroGFP1.1, and eroGFP1.2 in 0.1 mM diamide and 5 mM DTT.

[0032] Figure 5 BD represents the maximum response of roGFP1 (B), eroGFP1.1 (C), and eroGFP1.2 (D) at different pH values ​​(5.5–8.5).

[0033] Figure 6 A represents the maximum response of the mutant at different temperatures ranging from 25 to 40°C.

[0034] Figure 6 BC represents the dual-channel fluorescence of the redox sensor after heat shock at 60-90℃ for 30 min. F400 (F), F480 (G).

[0035] Figure 6 D represents the effect of chloride perturbation on the roGFP sensor response. Oxidized and reduced proteins were diluted in HEPES buffer (100 mM HEPES, pH 7.3) containing different amounts of NaCl. N = 3. Error bars represent SEM.

[0036] Figure 7 Representative images of HeLa cells expressing roGFP1 (top) and eroGFP1.2 (bottom) (F) 482 ).

[0037] Figure 8 HeLa cells expressing eroGFP1.2 were treated with 100 μM diamino group at 0 min, followed by 5 mM DTT at 15 min. The pseudo-color ratio images of cells at the time points shown illustrate the response kinetics of eroGFP1.2.

[0038] Figure 9 A represents the excitation rate-time progression of eroGFP1.2 in this experiment, plotted as the average of ten individual cells. The addition of diamino and DTT is indicated by arrows.

[0039] Figure 9 B represents the normalized F value of the eroGFP1.2 sensor in cells without interference (Ctrl), DTT treatment (reduction), or diamine treatment (oxidation). 407 / F 482 .

[0040] Figure 9 C represents the oxidation fraction of the cytoplasmic eroGFP1.2 sensor under control conditions.

[0041] Figure 9D represents the calculated cytoplasmic redox potential, approximately -328 mV. Scale bar, 10 μm.

[0042] Figure 10 A represents the fluorescence of roGFP1 and eroGFP1.2 in HeLa cells.

[0043] Figure 10 B represents the fluorescent traces of the response of 10 HeLa cells expressing cytoplasmic eroGFP1.2 to 100 μM diamide and 5 mM DTT.

[0044] Figure 10 C represents the maximum dynamic changes in roGFP1 and eroGFP1.2 in the cytoplasm of HeLa cells.

[0045] Figure 11 Targeting and calibration of the eroGFP1.2 sensor in different subcellular organelles.

[0046] Figure 11 AD is an image (F482) of eroGFP1.2 fluorescence targeting mitochondria (A), plasma membrane (B), Golgi apparatus (C), and endoplasmic reticulum (D). Scale bar, 10 μm.

[0047] Figure 11 E represents the oxidation fraction of redox sensors located in different subcellular compartments of HeLa cells. The experiment is similar to that of eroGFP1.2 in the cytoplasm.

[0048] Figure 11 F represents the redox balance level in seven organelles of HeLa cells estimated by fitting in vitro redox titration curves.

[0049] Figure 12 The fluorescence of the eroGFP1.2 sensor in different subcellular organelles.

[0050] Figure 12 AD compares the fluorescence intensity of eroGFP1.2 and roGFP1 in different compartments of HeLa cells. Mitochondrial matrix (A), plasma membrane (B), Golgi apparatus (C), endoplasmic reticulum (D).

[0051] Figure 13 and Figure 14 To simultaneously visualize redox potential and H2O2 kinetics at the wound edge by co-expressing eroGFP1.2 and HyPerRed in zebrafish juveniles at 100 μM DPI or without DPI. The caudal fin was harvested at 0 min. Scale bar, 50 μm. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0053] Explanation of the relevant definitions in this invention: roGFP / roGFPs: This is a broad category of green fluorescent proteins that are genetically engineered and used to detect redox states within cells.

[0054] roGFP1: It is a member of the large class of green fluorescent proteins and is a prototype probe.

[0055] roGFP2 is a member of the large class of green fluorescent proteins. It is an improved member obtained through further gene mutation and screening of roGFP1.

[0056] eroGFP / eroGFPs: Endoplasmic Reticulum-targeted roGFP is an optimized roGFP2 gene sequence fused with an endoplasmic reticulum signal peptide and an endoplasmic reticulum retention signal. This allows it to be specifically expressed and retained in the endoplasmic reticulum lumen, thus enabling it to be used specifically for measuring the redox state within the endoplasmic reticulum.

[0057] sfGFP: or superfolder GFP, refers to protein that has been specially engineered to undergo mutations at superfolding sites (S30R / Y39N / N105T / Y145F / I171V / A206V) to improve folding efficiency, stability, and compatibility with fusion proteins.

[0058] This invention discloses an enhanced green fluorescent protein mutant, whose amino acid sequence has the following mutation sites compared with the amino acid sequence of green fluorescent protein roGFP1 or roGFP2: S30R, Y39N, N105T, I171V, A206V, L220F.

[0059] For ease of explanation, the enhanced green fluorescent protein mutant of this invention, compared with the amino acid sequence of the green fluorescent protein roGFP1, has the following mutation sites: S30R, Y39N, N105T, I171V, A206V, L220F, and is named eroGFP1.2. Its amino acid sequence is shown in SEQ ID No:1, and the schematic diagram is as follows. Figure 1A and 1B As shown.

[0060] Compared with the amino acid sequence of the green fluorescent protein roGFP2, the enhanced green fluorescent protein mutant with the following mutation sites S30R, Y39N, N105T, I171V, A206V, and L220F is named eroGFP2.2, and its amino acid sequence is shown in SEQ ID No:2. A schematic diagram is shown below. Figure 1C As shown.

[0061] The present invention also discloses a fluorescent probe, wherein the raw materials for preparing the fluorescent probe include the enhanced green fluorescent protein mutant eroGFP1.2 or eroGFP2.2 of the present invention.

[0062] This invention also discloses the use of enhanced green fluorescent protein mutants in oxidation sensors. The oxidation sensor has any of the following uses: a. monitoring redox balance dynamics, b. real-time redox potential calibration, c. in-situ imaging, d. visualization of cellular redox potentials.

[0063] A control group was designed during the study, as follows: (1). Control group 1: The amino acid sequence of green fluorescent protein roGFP1 was mutated to F46L and named roGFP1-F46L.

[0064] (2). Control group 2: The amino acid sequence of green fluorescent protein roGFP1 was mutated to M153T and named roGFP1-M153T.

[0065] (3). Control group 3: The amino acid sequence of green fluorescent protein roGFP1 was mutated by L220F and named eroGFP1.0.

[0066] (4). Control group 4: The amino acid sequence of green fluorescent protein roGFP1 was mutated by S30R, Y39N, N105T, I171V, A206V, Y145F, and L220F, and named eroGFP1.1. eroGFP1.2 lacks the Y145F mutation compared to eroGFP1.1.

[0067] (5). Control group 5: The amino acid sequence of green fluorescent protein roGFP2 was mutated by L220F and named eroGFP2.0.

[0068] (6). Control group 6: The amino acid sequence of green fluorescent protein roGFP2 was mutated to S30R, Y39N, N105T, I171V, A206V, Y145F, and L220F, and named eroGFP2.1. eroGFP2.2 lacks the Y145F mutation compared to eroGFP2.1.

[0069] To demonstrate the effectiveness of this invention, the following experiments were conducted for verification and illustration.

[0070] (I) Experimental Materials and Methods 1. Rational design and plasmid construction To generate mutants of the protein of this invention and mutants of the control group, the corresponding F46L, M153T, L220F variants and hyperfolding mutation sites (S30R / S39R / N105T / Y145F / I171V / A206V) were introduced into roGFP1 or roGFP2. All variants were constructed on the pRSETb vector using Prime Star DNA polymerase via reverse PCR. For cytoplasmic expression in HeLa cells, the cDNA encoding eroGFP1.2 was digested with BamHI and HindIII and ligated into pcDNA3.1(+). For expression of eroGFPs in the mitochondrial matrix, a double-stranded mitochondrial signal peptide of a cytochrome c oxidase subunit VIII was fused to the N-terminus. Other plasmids carrying specific subcellular signals were derived from Addgene, directly replacing the initial gene with an eroGFP sensor.

[0071] 2. Protein expression and purification The constructed plasmid was expressed in *E. coli* BL21(DE3) and induced with 1 mM isopropyl-β-thiogalactoside (IPTG). The recombinant protein was purified by nickel affinity chromatography (GE Healthcare) according to the manufacturer's protocol. Briefly, the cell pellet was resuspended in binding buffer (100 mM sodium phosphate, 500 mM sodium chloride, and 50 mM imidazole, pH 7.4) and sonicated for 10 min. Cell lysates were centrifuged at 9,000 g for 30 min and then applied to a pre-equilibrated Ni-NTA column, followed by washing with binding buffer and elution with buffer containing 300 mM imidazole. The purified protein was concentrated by filtration (Centricon 10; Amicon) and desalted to standard HEPES buffer (100 mM HEPES, 100 mM sodium chloride; pH 7.4) via (Sephadex G25, GE Healthcare).

[0072] 3. Measurement of fluorescence properties in vitro and in bacteria Fluorescence spectra were collected at 37°C using a Cary Eclipse fluorescence spectrometer (Varian). Samples consisted of 1 μM sensor, 10 mM DTT, and 0.1 mM diamine to obtain reduced and oxidized states. For redox titration, the probe was diluted in degassed HEPES buffer containing 1 μM protein and 10 mM DTT (a mixture of oxidized and reduced forms, in a ratio from 0:10 to 10:0, in increments of 1 mM) and incubated at 30°C for 1 h, after which fluorescence excitation intensity was measured. The thermal stability of the redox sensor was assessed by denaturation at 90°C for 30 min using a PCR machine (Biometra, Germany), and the brightness of the denatured samples was immediately collected using a microplate reader.

[0073] The optical density of roGFP-expressing bacteria was diluted to 0.1 with HEPES buffer, and fluorescence was measured in triplicate (excitation wavelengths of 420 nm and 485 nm, and emission wavelength of 528 nm). The reaction kinetics of the indicators were monitored by adding 10 mM DTT and 500 μM diamine to E. coli cell suspension for 30 minutes, and the dynamic range was calculated using the oxidation-reduction ratio.

[0074] 4. Cell culture and imaging HeLa cells were maintained in Dürbeck Modified Eagle Medium (DMEMhyclone) supplemented with 10% FBS (Gibco). Cells were digested and counted, then seeded at 40,000 cells per well in a 4-well glass dish (35 mm²). The plasmid encoding eroGFP1.2 was transfected using Liposome Transfection Reagent 2000 (Invitrogen) according to the manual instructions. Approximately 24 hours post-transfection, cells were imaged in HBSS buffer (10 mM HEPES, 140 mM NaCl, 5 mM KCl, 4 mM NaHCO3, 0.45 mM KH2PO4, 0.35 mM NaHPO4, 1 mM CaCl2, 0.5 mM MgSO4, 10 mM D-glucose, pH 7.4). Wide-field observation was performed using a PlanApoVC60, 1.20 numerical aperture (NA) water immersion objective on a Nikon Eclipse Ti-E automated microscope (Nikon Instruments).

[0075] The filters used for dual excitation ratio imaging of eroGFP1.2 were purchased from Semrock: a 407 / 17 excitation filter, a 482 / 30 excitation filter, and a 535 / 40 emission filter. These two excitation filters were used alternately via a Lambda10-XL filter wheel (Shutter Instruments). Images were captured in 1280×1024 format with 12-bit depth. A pseudo-color image was created by dividing the 407 nm excitation image by the 480 nm excitation image of the same cell using ImageJ software.

[0076] 5. In vitro transcription The coding sequences for enhanced redox-sensitive green fluorescent protein (eroGFP1.2) and the red hydrogen peroxide sensor (HyPerRed) were cloned into the pTol2 vector backbone containing the T7 promoter and SV40 polyadenylation signal. Capped mRNAs were synthesized in vitro using the mMESSAGEmMACHINE kit (Qiagen) following the manufacturer's instructions after PCR amplification. Residual DNA template was digested with 2 units of DNase I, and the mRNA was purified by precipitation with lithium chloride at -20°C for 2 hours.

[0077] 6. Zebrafish rearing and internal imaging Zebrafish (Danio rerio) were reared under standard laboratory conditions according to established protocols. For dual imaging of H2O2 and redox dynamics, eroGFP1.2 and HyPerRed mRNA were mixed at equal concentrations (final concentration: 50 ng / μl each). One nanoliter of the mixture was injected into single-cell stage zebrafish embryos. Post-injection embryos were cultured in egg water at 28°C, supplemented with 0.2 mM N-phenylthiourea (PTU; Sigma) to inhibit melanin production and ensure optical transparency. Embryos were reared for 2 days under a standard light / dark cycle prior to imaging experiments. Two days post-fertilization (dpf), larvae were anesthetized in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, 0.6 mM Tricaine). The tip of the caudal fin was removed using a scalpel or microsurgical blade under a stereomicroscope. After being injured, the larvae were embedded in 1% low-melting-point agarose and placed in a glass-bottomed petri dish for subsequent imaging.

[0078] To assess the contribution of NADPH oxidase to ROS production, larvae were pretreated in E3 medium with 0.1 mM diphenyl iodide (DPI; Sigma) for 30 min prior to tail fin injury, as previously described. In vivo imaging was performed using a fluorescence microscope equipped with a Plan Apo 20× / 0.75 NA objective. The following excitation / emission filter sets were used: eroGFP1.2 excitation: 407 / 17 nm or 482 / 35 nm; emission: 535 / 40 nm; HyPerRed excitation: 560 / 40 nm; emission: 630 / 60 nm. Image processing and quantitative analysis were performed using the standardized methods described previously. All settings remained constant throughout the imaging process to ensure consistency of experimental conditions.

[0079] 7. Statistics and Repeatability Two-way ANOVA was used for paired comparisons. Paired Student's t-tests were used for between-group comparisons. Sample sizes were not predetermined using statistical methods. All experiments were replicated from independent experimental units or biological replicates of subjects. Error bars or shaded areas for all data represent standard errors (SEM). For statistical analyses, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. All statistical analyses and graphical representations were performed using Graphpad Prim (V9.5.1) or Sigmaplot 12.5. Figures were compiled and finalized using Adobe Illustrator 2020. Schematic diagrams were created using BioRender.

[0080] (II) Experimental Results 1. Fluorescence properties like Figure 2 As shown in 2B and 2C, eroGFP1.0 (L220F mutation) increases the maximum dynamic range by 1.6 times; however, the fluorescence intensity is reduced by half compared to the parent roGFP1. It is speculated that the large aromatic amino acids may interact with the pigment to amplify the reaction, but may also impair protein folding.

[0081] like Figure 2 As shown in B and 2D, the baseline fluorescence of the mutants eroGFP1.1 and eroGFP1.2 was increased by approximately 3.5-fold and 5-fold, respectively, compared to roGFP1. Furthermore, the dynamic range was increased by approximately 50% compared to roGFP1 expressed in bacteria. Figure 2 C).

[0082] like Figure 3As shown in D, compared with roGFP1, the fluorescence effects of roGFP1 sensors with different superfolder mutations expressed in bacteria vary. The effect of a single mutation is not as good as the effect of mutations at 6 mutation sites, while the fluorescence effect of only 5 mutations (omitting the Y145F mutation) is worse than that of 6 mutations.

[0083] like Figure 3 As indicated by 3F and 3G, eroGFP2.0 shows a decrease in both fluorescence intensity and response compared to roGFP2. eroGFP2.1 shows a slight increase in fluorescence intensity compared to roGFP2.0, but the increase is not significant. eroGFP2.2, however, shows a significant increase in fluorescence intensity compared to roGFP2.0. This is because the Y145F mutation reduces polarity, impairing chloroplast maturation and thus reducing fluorescence, similar to the situation observed in eroGFP1.1.

[0084] like Figure 4 As shown in Figure 4A, the fluorescence spectrum of eroGFP is very similar to that of its parent roGFP1, exhibiting two excitation peaks near 400 nm and 480 nm, and a common emission peak at approximately 510 nm. After oxidation, the excitation intensity of the reduced form of eroGFP decreases more than fourfold at 480 nm, while increasing by 30% at 400 nm. This is due to the excitation ratio R... 400 / 485 The defined dynamic range reached approximately 6.0 times for eroGFP1.1 and approximately 6.5 times for eroGFP1.2. Figure 4 B, Figure 5 A).

[0085] Redox titration further revealed that the midpoint redox potential of eroGFP1.1 was -264 mV, and that of eroGFP1.2 was -266 mV. Figure 4 C), which is slightly more oxidizing than the potential of roGFP1 (-285 mV). Notably, the fluorescence excitation ratio of eroGFP remains stable between pH 5.5 and 8.5. Figure 4 D, Figure 5 B- Figure 5 (D), confirming its robustness to physiological pH fluctuations.

[0086] Considering that the model organism can survive over a wide temperature range, the temperature dependence of the roGFP sensor response was further evaluated in the experiments. The maximum response remained constant between 25°C and 40°C. Figure 6A). Notably, we observed that the roGFP sensor exhibited resistance at high temperatures ranging from 60°C to 90°C. Under these extreme conditions, eroGFP1.2 retained approximately 40% of its fluorescence signal after 30 minutes at 90°C, while roGFP1 retained only approximately 20%. Figure 4 E, Figure 6 B, Figure 6 C), which indicates that its thermal stability has been improved.

[0087] The experiment also investigated the effect of chloride ion concentration on sensor performance. The fluorescence emission ratio of eroGFP remained essentially unchanged under different chloride ion concentrations. Figure 6 (D), which indicates that these sensors can be used for a variety of subcellular organelles.

[0088] 2. Imaging of cytoplasmic redox changes Figures 7-10 This provides experimental data for monitoring the dynamics and kinetics of redox reactions in the cytoplasm of HeLa cells. To determine the function of eroGFP1.2 in mammalian cells, it was subcloned into the pcDNA3.1 vector and expressed in HeLa cells. After expression, eroGFP1.2 exhibited strong fluorescence throughout the cell, such as... Figure 7 As shown. Quantitative analysis revealed that the fluorescence intensity of eroGFP1.2 (F407 and F482) was significantly higher than that of the original roGFP1, approximately 6 times higher. Figure 10 A). Fluorescence ratio imaging (F407 / F482) showed no significant difference in signal between the cytoplasm and the nucleus. Figure 8 This indicates that the basic redox potentials are similar in these compartments.

[0089] The baseline fluorescence of eroGFP1.2 positive cells was monitored in the experiment for 3 minutes. Figure 8 , Figure 9 A and Figure 10 B). When 100 μM diamine was applied, a strong oxidizing agent, the cells exhibited a rapid and intense oxidation response, with fluorescence increasing approximately 3-fold within 2 minutes. Figure 8 , Figure 9 A and Figure 10 C). Subsequently, using 5 mM dithiothreitol (DTT), a reducing agent, effectively reversed the oxidation sensor back to a reduced state, even below the initial baseline state. Figure 8 , Figure 9 B and Figure 10 B). The response of eroGFP1.2 was approximately 1.7 times higher than that of roGFP1, demonstrating an improved dynamic range in mammalian cells (B). Figure 10 C).

[0090] To characterize the redox potential in the cytoplasm, the excitation ratio in cells was set to a minimum and a maximum ratio under treatment with a reducing agent (5 mM DTT) and an oxidizing agent (100 μM diamide). Figure 9 B and Figure 10 C). It can be observed that the oxidation fraction of cytoplasmic sensors in HeLa cells under physiological conditions is approximately 8.3% ( Figure 9 C), which means that after fitting the in vitro calibration curve, the baseline redox potential is -328 mV ( Figure 9 D).

[0091] These findings demonstrate that eroGFP1.2 works effectively in living mammalian cells, enabling dynamic and reversible monitoring of intracellular redox changes. Its enhanced fluorescence intensity and quantitative response make it a valuable tool for real-time redox potential calibration and in-situ imaging.

[0092] 3. Calibration of intracellular redox potential in HeLa cells Further experiments investigated whether enhanced eroGFP1.2 could detect redox potentials in specific subcellular compartments of mammalian cells. eroGFP1.2 was targeted to multiple organelles, including the mitochondrial matrix, plasma membrane (PM), Golgi apparatus (Gol), and endoplasmic reticulum (ER), using validated localization signals. Specific target signals for each compartment were obtained from the non-profit plasmid library Addgene. Figure 11 The targeting and calibration of the eroGFP1.2 sensor in different subcellular organelles were demonstrated, and fluorescence imaging confirmed the expected localization pattern of each construct. Figure 12 The fluorescence of the eroGFP1.2 sensor in different subcellular organelles was expressed.

[0093] Comparative analysis showed that, in all targeting compartments, the fluorescence intensity of eroGFP1.2 was significantly higher than that of roGFP1 ( Figure 12 (AD). Specifically, eroGFP1.2 showed at least a 5-fold increase in brightness in mitochondria, endoplasmic reticulum, and plasma membrane. In the Golgi apparatus ( Figure 12 A, Figure 12 B and Figure 12 The fluorescence intensity of eroGFP1.2 is approximately 2.5 times that of roGFP1 (D). Figure 12 C).

[0094] Next, redox calibration was performed in each compartment using the same in-situ titration protocol established for the cytoplasm. Figure 11 E, Figure 11F). eroGFP1.2 located in the endoplasmic reticulum was almost completely oxidized (approximately 97%), indicating a strongly oxidizing environment (-221 mV). The Golgi apparatus showed an oxidation level of approximately 70%, with a redox potential of -253 mV. The plasma membrane exhibited an oxidation state similar to the cytoplasm, with an oxidation percentage of 9.5% and a potential of -324 mV. The mitochondrial matrix maintained a more reduced redox state (-350 mV) than other subcellular organelles. This observation is consistent with previous findings and supports the reliability of eroGFP1.2 in compartment redox mapping.

[0095] These findings suggest that the high signal intensity of eroGFP1.2 enables it to effectively target, visualize, and quantify different redox potentials at multiple subcellular locations in mammalian cells.

[0096] 4. Visualization of redox changes in zebrafish tissue To achieve in vivo visualization of reductive-oxidative changes, mRNAs encoding eroGFP1.2 and HyPerRed (a red fluorescent H2O2 sensor) were co-injected into single-cell stage zebrafish embryos. These two sensors were effectively co-expressed ( Figure 13 , Figure 14 Furthermore, at 2 days post-fertilization (dpf), the fluorescent signal in zebrafish larvae was clearly visible. Following caudal fin injury, eroGFP1.2 showed significant cytoplasmic oxidation at the wound edge within 25 minutes. Simultaneously, the HyPerRed signal intensity increased significantly, demonstrating precise spatial and temporal colocalization with the eroGFP1.2 oxidation signal. Figure 13 ).

[0097] These observations indicate that tissue damage rapidly reduces intracellular NADPH levels, thereby promoting H2O2 production through NADPH oxidase activity. To further verify this mechanism, larvae were pretreated with diphenyl iodide (DPI), a selective NADPH oxidase inhibitor. DPI treatment completely eliminated cytoplasmic oxidation detected by eroGFP1.2 and the increase in H2O2 at the wound site quantified by HyPerRed. Figure 14 This confirms that the redox reaction depends on the NADPH oxidase.

[0098] Overall, these results establish eroGFP1.2 as a robust and reliable sensor, particularly in the context of damage-induced oxidative signaling, for capturing dynamic intracellular redox changes in vivo.

[0099] Through the above experimental studies, the enhanced green fluorescent protein provided by this invention exhibits significantly enhanced brightness (approximately 5 times higher than roGFP1) and expanded dynamic range (approximately 50%). This enables quantitative spatiotemporal imaging of redox dynamics in mammalian cells and zebrafish. The intrinsic ratiometric properties of eroGFP1.2 minimize artifacts caused by variations in cell thickness, photobleaching, or expression levels, thereby achieving robust and reliable measurements in a variety of biological contexts.

[0100] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. An enhanced green fluorescent protein mutant, characterized in that: Compared with the amino acid sequences of green fluorescent proteins roGFP1 or roGFP2, its amino acid sequence has the following mutation sites: S30R, Y39N, N105T, I171V, A206V, L220F.

2. The enhanced green fluorescent protein mutant as described in claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID No:1 or SEQ ID NO:

2.

3. A fluorescent probe, characterized in that: The raw materials for preparing the fluorescent probe include the enhanced green fluorescent protein mutant as described in claim 1 or 2.

4. The method for preparing the enhanced green fluorescent protein mutant according to claim 1 or 2, characterized in that, Includes the following processes: Mutations were introduced at the mutation sites in green fluorescent protein roGFP1 or roGFP2: serine at position 30 was mutated to arginine, tyrosine at position 39 to asparagine, asparagine at position 105 to threonine, isoleucine at position 171 to valine, alanine at position 206 to valine, and leucine at position 220 to phenylalanine. All mutations were constructed using Prime Star DNA polymerase on the pRSETb vector via reverse PCR.

5. The method for preparing the enhanced green fluorescent protein mutant as described in claim 4, characterized in that, The constructed plasmid was expressed in Escherichia coli and induced to form a recombinant protein via isopropyl-β-thiogalactoside.

6. Use of the enhanced green fluorescent protein mutant of claim 1 or 2 in an oxidation sensor.

7. The use as described in claim 6, characterized in that: The oxidation sensor has any of the following uses: a. Monitor the dynamics of redox balance; c. Real-time redox potential calibration; d. In-situ imaging of subcellular organelles and detection of their redox potential; e. Visualize the redox potential of cells.

Images