The Bradford assay is a widely used and reliable method for determining the protein concentration in biological samples. This method was developed by Marion M. Bradford in 1976 and is renowned for its simplicity and high sensitivity. This colorimetric technique is an important foundation in biochemistry and molecular biology research. By taking advantage of the unique interaction between Coomassie Brilliant Blue dye and proteins, the Bradford assay can quickly and accurately estimate protein levels, even in complex mixtures (Bradford, 1976).

The principle of the Bradford assay is based on the differential binding of the Coomassie Brilliant Blue G-250 dye to proteins under acidic conditions, which results in a shift in the maximum absorption wavelength. In the unbound state, this dye exists in a cationic red form, with its absorption peak at 465 nanometers. When it binds to proteins (mainly through non-covalent interactions with basic (such as arginine, lysine) and aromatic amino acid residues (such as tryptophan, tyrosine)), the dye transforms into a stable anionic blue form, which has a distinct absorption peak at 595 nanometers. The intensity of this blue color (measured by spectrophotometry at 595 nanometers) is proportional to the protein concentration in the sample (Bradford, 1976) For each experiment, we set up three parallel runs to ensure the stability of the detection, and use PBS solution as the blank control to ensure the accuracy of the detection..

1. Material: BSA standard solution, Bradford solution, PBS, 96-well plate
2. Equipment: Microplate reader, multi-channel pipettes

• Preparation:

1. Equilibrate all reagents to room temperature.
2. Prepare BSA standard dilutions using the same buffer as samples.

Table 1 Dilution system of BSA standard substances

• Standard Curve Setup

1. Add 5 μL of each BSA standard or sample to a 96-well plate.
2. Add 250 μL of Bradford solution to each well. 

• Incubation

Incubate at room temperature for 5 minutes (avoid exceeding 1 hour).

• Measurement

1. Read absorbance at 595 nm using a microplate reader.
2. Generate a standard curve by plotting absorbance vs. BSA concentration.
3. Calculate sample protein concentration from the standard curve equation

Protein concentrations of both purified native allergens and their hypoallergenic derivatives were quantified using the Bradford assay to ensure accurate loading for subsequent IgE-binding affinity assays. A standard curve was constructed using bovine serum albumin (BSA) as a reference, exhibiting excellent linearity (R² = 0.9987) with the regression equation Y = 0.001563x + 0.002397 (Figure 1). All absorbance measurements at 595 nm were performed in triplicate, and sample concentrations were interpolated based on the standard curve (Table 2).

Figure 1 BSA standard curve test by Bradford assay. (A) The absorbance values of the BSA standard samples. (B) Fitting results of the standard curve.

Table 2 Protein quantification results.

Accurate protein quantification is of vital importance for subsequent functional analysis, as it ensures the comparability and reproducibility of the experiments. We conducted a reliable protein concentration determination using the Bradford assay. This can be clearly seen from the excellent linearity of the standard curve (R² = 0.9987) and the extremely small errors between the parallel experiments.

To enhance the reproducibility of this experiment, we have compiled a troubleshooting guide based on the issues encountered during the experimental process, providing assistance to the iGEM community.

1. The standard curve shows poor linearity.

 Possible Cause-1: The protein standard was degraded or the preparation was incorrect.

 Solution-1: Prepare fresh standard solutions, aliquot and freeze them to avoid repeated freezing and thawing. Dilute the sample to its optimal dynamic range (usually 1 - 200 μg/mL) to achieve the best linear range.

 Possible Cause-2: There were significant differences in the incubation time for each well after the dye was added.

 Solution-2: Use multi-channel pipettes to quickly add the dye and immediately shake the plate to mix.

2. The repeatability of the sample is poor.

 Possible Cause-1: The samples were not thoroughly mixed.

 Solution-1: Before the experiment, vortex shaking or pipetting should be used to mix all the samples and standards evenly.

 Possible Cause-2: After adding the sample, there are still some bubbles remaining on the liquid surface.

Solution-2: Remove the bubbles on the liquid surface through centrifugation.

We conducted IgE binding affinity analysis, the main purpose of which is to functionally evaluate the allergenic potential of natural and modified hypoallergenic derivatives, and fusion hypoallergenic derivatives. This assessment is crucial because the clinical severity of allergic reactions is largely determined by the affinity of the specific IgE antibodies against the allergen and its target epitope (Fromberg, 2006). High-affinity IgE antibodies can trigger strong activation of mast cells and basophils even in the presence of low concentrations of allergens, leading to significant release of inflammatory mediators and potential anaphylactic shock. In contrast, low-affinity IgE has significantly reduced ability to bind to the FcεRI receptor, thereby reducing the risk of severe allergic reactions (Udoye, Ehlers, & Manz, 2023). Therefore, by quantifying the degree of reduction in IgE binding affinity, it can provide a key indicator for in vitro experiments to determine whether our rational protein design strategy has successfully achieved the reduction of allergenicity.

The traditional immunoblotting technique, non-denaturing dot blotting, can effectively demonstrate the differences in the binding of low-reactive antigens to IgE compared to natural antigens (Chen, 2012). However, it has some key limitations: it is semi-quantitative in nature, inefficient, and relies on radioactive labeling (such as ¹²⁵I), which poses challenges in terms of safety and standardization. In contrast, ELISA can perform true quantitative analysis through a standard curve, support high-throughput screening in microplates, and adopt more convenient detection methods (such as enzyme-linked color development/chemiluminescence signals), providing better safety and accuracy. Therefore, although immunoblotting can still be used for preliminary verification, ELISA has more outstanding advantages in conducting strict and reproducible quantitative analysis of IgE affinity. Quantitative determination of the reduction in IgE binding affinity can serve as an in vitro indicator for evaluating the effectiveness of allergens in reducing allergies.

We designed an in vitro ELISA method to assess the ability of our designed protein to bind to allergens, comparing it with their natural forms. In brief, the microplate was first coated with purified natural proteins or hypoallergenic derivatives. Then, it combined with IgE antibodies. The binding of IgE was detected using a labeled anti-human IgE antibody. The enzymatic reaction generated a colorimetric or chemiluminescent signal proportional to the amount of bound IgE. The intensity of this signal was compared between different samples, allowing for the precise calculation of the binding rate of each hypoallergenic derivative to IgE. For each experiment, we set up three parallel runs to ensure the stability of the detection. We used natural allergens (prepared by ourselves or purchased) as positive controls, and PBS solution as the blank control to ensure the accuracy of the detection.

• PBS (0.1 M, pH 7.4)
• 96-well plates (High Binding)
• The sample to be tested (allergen protein)
• Wash buffer (PBS containing 0.05% Tween-20)
• Blocking buffer (5% BSA in PBS)
• Closure plate membrane
• Pre-coated micro well plates
• IgE standard
• Standard/Sample diluent (SD1)
• Concentrated Biotin-conjugated IgE(Immunoglobulin E) antibody (100×)
• Biotin-conjugated antibody diluent (SD2)
• Concentrated HRP-conjugated Streptavidin (100×)
• HRP-conjugated Streptavidin diluent (SD3)
• Substrate reagent (TMB solution)
• Stop Solution (2M H₂SO₄)

• Protein-Coated 96-Well Plate

1. Dilute the allergen protein to a concentration of 10 μg/mL using PBS (pH 7.2).
2. Add 0.1 mL of the diluted allergen protein solution to each well of an ELISA plate, and incubate overnight at 4°C.

• Blocking

1. Add 100 µL of blocking buffer (1% BSA in PBS) to each well.
2. Incubate at 37°C for 1 hour.
3. Wash the wells to remove unbound blocking reagent.

• Preparation of IgE Standards

1. Centrifuge the lyophilized IgE standard at 10,000 × g for 1 minute.
2. Reconstitute by adding 1.0 mL of sample dilution buffer, invert gently, and allow to dissolve for 10 minutes. Mix thoroughly.
3. Prepare a 200 ng/mL stock solution, then perform serial two-fold dilutions to obtain the following concentrations: 200, 100, 50, 25, 12.5, 6.25, 3.13, 0 ng/mL.
4. Use 7 tubes, each preloaded with 500 µL dilution buffer. Add 500 µL from the previous tube, mix well, and continue stepwise. The final tube (0 ng/mL) serves as the blank. Add the diluted sample to the 96-well plate.

• IgE Binding to Allergen-Coated Wells

1. Add 100 µL of IgE working solution (50 ng/mL) to each well. For the standard curve, use the full dilution series.

Figure 2 Layout of ELISA sample addition.

2. Incubate at 37°C for 90 minutes.
3. Note: Add samples gently to the center of the well, avoiding the walls; mix gently without bubbles. Complete sample loading within 10 minutes.
4. Prepare biotin-labeled anti-IgE antibody by diluting the 100× stock 1:100 in SD2 buffer (calculate required volume based on 100 µL per well). Use within 30 minutes.
5. Discard the well contents and tap dry. Add 100 µL of the biotin-labeled antibody to each well.
6. Incubate at 37°C for 60 minutes, then wash 4 times with 350 µL wash buffer (soak 1–2 minutes per wash).

• Streptavidin-HRP Conjugate Binding

1. Dilute the 100× HRP-labeled streptavidin stock 1:100 in SD3 buffer (post-centrifugation), based on 100 µL/well. Use within 30 minutes.
2. Add 100 µL of the HRP conjugate to each well.
3. Incubate at 37°C for 30 minutes, then wash 4 times using 300 µL per wash (30 s intervals).

• Color Development

1. Add 90 µL of TMB substrate to each well (protect from light).
2. Incubate at 37°C in the dark for ~15 minutes.

• Reaction Termination

Add 50 µL stop solution to each well and immediately read absorbance at 450 nm using a microplate reader (within 5 minutes).

• Data Analysis

1. Subtract the OD value of the blank (0 ng/mL) from all standards and sample readings.
2. Plot the standard curve using IgE concentrations on the X-axis and corresponding OD values on the Y-axis.
3. Fit the curve using a four-parameter logistic regression model to quantify IgE binding in unknown samples.

The standard curve for human IgE quantification was generated using four-parameter logistic (4PL) regression in GraphPad Prism 9.5, demonstrating excellent fit (R² > 0.998), which validated the reliability of subsequent affinity measurements (Figure 3A). The IgE-binding capacities of six purified allergens—both native and hypoallergenic derivatives—were evaluated by ELISA. Coated proteins were incubated with 50 ng/mL human IgE. As shown in Figure 3B, significant reduction in IgE binding (p < 0.0001) was observed in four hypoallergenic derivatives compared to their native counterparts (mBet v1, mDer p2, mMet e1, mCyp c1). Although mPhl p1 and mDer p1 also showed decreased affinity, the reduction was more modest (p>0.05). These results confirmed the effectiveness of the rational design strategy in generating hypoallergenic proteins with markedly reduced IgE reactivity.

Table 3 The calculation results of IgE binding rate of single allergen.

Figure 3 IgE affinity of allergen proteins. (A) IgE standard curve, (B) Comparison of natural allergens and hypoallergenic derivatives.

As illustrated in Figure 4A, the IgE standard curve demonstrated excellent linearity (R² = 0.9943), ensuring the reliability of the subsequent binding affinity quantification. Interestingly, comparative analysis revealed that two fusion allergens, ​​EFK8-mPhl p1-GS-mBet v1 (FPB)​​ and ​​EFK8-mDer p1-GS-mDer p2 (FDD)​​, exhibited significantly reduced IgE binding ratios compared to their respective single hypoallergenic constituents (mPhl p1 and mDer p1, respectively). ​​EFK8-mMet e1-GS-mCyp c1 (FMC)​​ also showed low IgE binding affinity, which was comparable to the levels observed for its individual hypoallergenic derivatives (mMet e1 and mCyp c1) (Figure 4B). This consistent reduction across all fusion constructs suggests that the fusion strategy effectively attenuates IgE immunoreactivity beyond the level achieved by individual engineered allergens, likely due to structural masking of key epitopes or altered antigen presentation.

Table 4 The calculation results of IgE binding rate of single and fusion allergens.

Figure 4 IgE affinity of allergen proteins. (A) IgE standard curve, (B) Comparison of single and fusion hypoallergenic derivatives.

The hypoallergenic derivatives after engineering modification showed a significantly lower IgE binding affinity compared to that observed in the natural allergen, indicating that our rational protein design strategy has achieved a remarkable overall effect in reducing allergenicity. Moreover, the IgE reactivity of the fusion proteins is lower than that of their respective hypoallergenic derivatives components, which may be attributed to the structural and conformational changes caused by fusion and oligomerization. These results confirmed the potential application of hypoallergenic derivatives based on fusion technology in the next generation of allergen immunotherapy (AIT), which possesses both broader coverage and lower risk of sensitization.

The hypoallergenic derivatives after engineering modification showed a significantly lower IgE binding affinity compared to that observed in the natural allergen, indicating that our rational protein design strategy has achieved a remarkable overall effect in reducing allergenicity. Moreover, the IgE reactivity of the fusion proteins is lower than that of their respective hypoallergenic derivatives components, which may be attributed to the structural and conformational changes caused by fusion and oligomerization. The addition of self-assembling peptides (EFK8) promotes the formation of higher-order complexes, which may spatially shield key IgE epitopes and disrupt the geometric integrity of the conformational epitopes that are crucial for high-affinity IgE cross-linking. These results confirmed the potential application of hypoallergenic derivatives based on fusion technology in the next generation of allergen immunotherapy (AIT), which possesses both broader coverage and lower risk of sensitization.

In the development of the AIT vaccine, evaluating its cell proliferation activity and biological safety is a crucial link that connects the in vitro potency verification and in vivo application. This step is of utmost importance as it directly affects the reliability and translational potential of the candidate vaccine during the preclinical research stage. If a vaccine effectively reduces the IgE binding affinity (i.e., low allergenicity) while not significantly inhibiting cell viability, it indicates good therapeutic potential, providing important safety basis for subsequent animal experiments and clinical applications.

The CCK-8 method, as a sensitive, reliable, and high-throughput cell viability detection method, is highly suitable for this assessment. This method detects the activity of dehydrogenase in the mitochondria of live cells, quantitatively reflecting the proliferation and metabolic status of the cells. Its results have a good linear relationship with the number of live cells, and can accurately identify excellent candidate vaccines that maintain low allergenicity and have no adverse effects on cell growth.

The CCK-8 (Cell Counting Kit-8) assay operates on the principle that ​​WST-8​​ (a water-soluble tetrazolium salt) is reduced by ​​cellular dehydrogenases​​ in metabolically active cells to generate an ​​orange-yellow formazan dye​​. This reduction occurs primarily in the mitochondria of viable cells and is facilitated by an electron carrier (e.g., 1-Methoxy PMS). The amount of formazan produced is ​​directly proportional to the number of living cells​​. The absorbance of the formazan solution is measured spectrophotometrically at ​​450 nm​​, providing a quantitative estimate of cell proliferation. The assay is highly sensitive, non-radioactive, and compatible with high-throughput screening. For each experiment, we set up three parallel runs to ensure the stability of the detection, and use PBS solution as the blank control to ensure the accuracy of the detection. NIH/3T3 cells (mouse embryonic fibroblasts) exhibit excellent repeatability and quantifiable metabolic activity in the CCK-8 assay due to their high contact inhibition properties and stable adherent growth capabilities. They can sensitively reflect changes in cell proliferation or toxicity, and are commonly used as a reliable model in drug screening and toxicity testing.

• NIH/3T3 cells
• DMEM complete medium
• 1× DPBS
• 0.25% trypsin solution
• T75 cell culture flasks
• 15 mL centrifuge tubes
• Hypoallergenic derivative, natural allergens, allergen fusion protein
• CCK-8

• Biosafety cabinet
• Inverted microscope
• CO₂ incubator (37 °C, 5% CO₂)
• Centrifuge (200 ×g)
• Pipettes and sterile tips

• Cell Revival

1. Rapidly take out frozen NIH-3T3 cells from liquid nitrogen.
2. Thaw the vial in a 37 °C water bath with gentle shaking. Do not exceed 1.5 min.
3. In a biosafety cabinet, slowly add the 1 mL thawed cell suspension to 9 mL pre-warmed complete DMEM medium in a centrifuge tube.
4. Mix gently by pipetting up and down.
5. Centrifuge at 200 ×g for 5 min.
6. Discard the supernatant carefully.
7. Resuspend the cell pellet in 1 mL of fresh complete DMEM medium.
8. Transfer the suspension into a T75 flask containing 9 mL complete DMEM medium.
9. Mix gently by crosswise shaking and place the flask into a 37 °C, 5% CO₂ incubator.
10. Replace medium with fresh complete medium on the second day, then every other day.

• Cell Passaging:

1. Observe cells under an inverted microscope; proceed when ~80% confluent.
2. In biosafety cabinet, discard old medium.
3. Add 3 mL DPBS, gently shake, discard; repeat wash once.
4. Add 1 mL of 0.25% trypsin along the flask wall (opposite to cells).
5. Incubate at 37 °C, 5% CO₂ for ~2 min.
6. Add 3 mL complete medium to stop digestion, gently shake to detach cells.
7. Pipette up and down to disperse cell clumps.
8. Transfer to centrifuge tube, spin at 200 ×g for 5 min, discard supernatant.
9. Resuspend pellet in 2 mL complete medium.
10. Add 9 mL fresh medium to a new T75 flask; seed appropriate volume of cell suspension.
11. Mix crosswise and place in incubator (37 °C, 5% CO₂).

• Cell Proliferation Test

1. Observe cells under an inverted microscope; proceed when ~80% confluent.
2. In biosafety cabinet, discard old medium.
3. Add 3 mL DPBS, gently shake, discard; repeat wash once.
4. Add 1 mL of 0.25% trypsin along the flask wall (opposite to cells).
5. Incubate at 37 °C, 5% CO₂ for ~2 min.
6. Add 3 mL complete medium to stop digestion, gently shake to detach cells.
7. Prepare a cell suspension with a density of 1×10⁵ cells/mL using complete medium. Transfer 90 μL of the cell suspension into a 96-well plate.
8. Preparation of drugs: Dilute each sample according to the table 5 to 1000, 100, and 10 nM (the concentration after adding to the cells will be 100, 10, and 1 nM respectively). Add 10 μL of drug solutions at different concentrations to a 96-well cell culture plate.

Table 5 Dilution of the test sample.

9. Add an equal volume of DPBS solution to the outer perimeter of the 96-well plate to prevent excessive evaporation.
10. After 24 h and 48h of incubation, add 10μL CCK-8 reagent to each well. Incubate at 37°C with 5% CO₂ for 30 min, then measure the absorbance at 450 nm using a microplate reader.

Figure 5 Layout of cell culture plate and drug treatment.

(11) Calculate cell viability using the following formula:

Cell viability (%) = P_e / P_b × 100%

P_e represents the absorbance of the experimental group and P_b represents the absorbance of the control group.

Control group: Cells + PBS + complete medium + CCK-8

Blank group: Complete medium + CCK-8

Experimental group: Cells + drug + complete medium + CCK-8

We evaluated the cytotoxicity of both single and fusion hypoallergenic derivatives using a CCK-8 assay to assess their biosafety for potential in vivo therapeutic applications. This evaluation is essential to ensure that engineered biologics exhibit minimal adverse effects on cell viability before proceeding to animal studies or clinical use.

First, we analyzed the cytotoxicity of individual hypoallergenic derivatives. For mBet v1 and mCyp c1, at all tested concentrations and time points, there was almost no significant effect on cell viability, emonstrating extremely high biocompatibility (Figure 6B, H). For mPhl p1 and mMet e1, the toxicity showed a clear time and concentration dependence (Figure 6A, G). At low concentrations and short durations, the impact was minimal, but as the concentrations and durations increased, the toxicity significantly intensified. For mDer p1 and mDer p2, even at lower concentrations and shorter durations, it could cause a significant decrease in cell viability, indicating an inherent toxicity (Figure 6D, E).

Next, we evaluated the cytotoxicity profiles of both individual and fused hypoallergenic derivatives. For the fusion construct EFK8-PT-mPhl p1-GS-mBet v1, we observed that the intrinsic cytotoxicity of mPhl p1 influenced the overall profile of the fusion protein, leading to a detectable reduction in cell viability particularly under high-concentration or prolonged exposure conditions (Figure 6A-C).In contrast, the fusion protein EFK8-PT-mMet e1-GS-mCyp c1 exhibited a different behavior: although mMet e1 alone showed time- and concentration-dependent cytotoxicity, the fusion with mCyp c1—which itself displayed no appreciable toxicity—resulted in a composite molecule that did not induce significant cytotoxicity, even at high doses or after extended incubation (Figure 6D-F). Notably, the fusion protein EFK8-PT-mDer p1-GS-mDer p2 did not exhibit significant cytotoxicity under the tested conditions. This is in clear contrast to its individual components, both mDer p1 and mDer p2, which caused a marked decrease in cell viability, especially at higher concentrations (e.g., 100 nM) and with longer culture periods (48 h) (Figure 6G-I). These results suggest that the fusion strategy can alter the cytotoxic properties of the constituent hypoallergenic derivatives, in some cases mitigating the adverse effects observed with the individual components.

Table 6 Relative cell viability calculation results.

Figure 6 The results of the cell proliferation experiment.

The cytotoxicity test results of CCK-8 indicated that both the individual hypoallergenic derivatives and their fusion constructs possess excellent biological safety characteristics, which are the key prerequisites for their further development as therapeutic vaccines. Particularly in the fusion protein EFK8-PT-mDer p1-GS-mDer p2, the observed reduction in toxicity compared to the individual mDer p1 and mDer p2 suggests that the fusion strategy itself may bring additional safety advantages. The alleviation of adverse reactions may be attributed to the following factors:

(1) The oligomerization mediated by EFK8 may have changed the overall molecular conformation, thereby possibly masking the residual hydrophobic or reactive regions that may trigger undesired cellular responses.

(2) The size increase resulting from the fusion may have reduced non-specific cellular uptake or led to the impact of cytotoxicity.

These findings are consistent with the broader concepts in engineered immunotherapy vaccines (Focke-Tejkl & Valenta, 2012). This significantly enhanced biocompatibility proves that they can be applied to more complex in vivo models and future clinical applications, thereby achieving safer and more effective specific allergen immunotherapy.

The pharmacokinetic characteristics of therapeutic proteins, especially their circulating half-life in the blood, are key factors determining the dosing frequency, patient compliance, and clinical efficacy. Traditional allergen immunotherapy (AIT) usually requires frequent administration because low-molecular-weight proteins are rapidly cleared from the blood through renal filtration and proteolysis. Strategies to prolong the half-life include fusing with large-molecule carrier proteins (such as albumin or Fc fragments), or using self-assembling peptides that can promote oligomerization to increase the hydrodynamic radius and protect vulnerable regions susceptible to enzymatic degradation (Andersen et al., 2011). The self-assembling peptide EFK8 (FEFEFKFK) has shown good efficacy in promoting the formation of stable nanoscale or oligomeric structures, through a mechanism that combines hydrophobic interactions (mediated by phenylalanine residues) and electrostatic interactions (mediated by lysine residues)(Song et al., 2023). During the drug development process, we need to assess the stability of the target protein through in vitro experiments, which lays an important preliminary foundation for the subsequent determination of the half-life of the candidate drug.In this part, we aim to investigate whether the EFK8 fusion protein can significantly prolong the half-life of low-allergenic proteins, using enhanced green fluorescent protein (EGFP) as a model protein to simulate the behavior of engineered allergens under physiological-related conditions.

The half-life assessment was accomplished by culturing the EFK8-EGFP protein and the control EGFP protein in 37°C fetal bovine serum for 5 days to simulate the physiological conditions in vivo. The serum contains various proteases and nucleases, which can simulate the natural degradation pathways. The integrity and concentration of EGFP were monitored by measuring the fluorescence intensity (excitation/emission: 488/507 nanometers), as the fluorescence properties of EGFP are directly related to its structural integrity. The fluorescence decay data were fitted using a one-phase exponential decay model. This method ensures accurate quantification of protein stability without interference from serum components, as fluorescence is only related to the functionally folded EGFP. For each experiment, we set up three parallel runs to ensure the stability of the detection, and use PBS solution as the blank control to ensure the accuracy of the detection.

• ​Purified ​​EFK8-EGFP​​ fusion protein (experimental group)
• Purified ​​non-fused EGFP​​ (control group)
• ​​Fetal bovine serum (FBS), sterile-filtered
• PBS (pH 7.4) for dilution

• Thermostatic incubator (37°C)
• Microplate reader
• Black 96-well plates (to minimize background fluorescence).
• Pipettes and sterile tips.

• ​​Sample Preparation​​:

1. Dilute both ​​FEFKFEFK-EGFP​​ and ​​non-fused EGFP​​ to ​​500 µg/mL​​ in FBS.
2. Aliquot ​​200 µL​​ of each protein-serum mixture into wells of a 96-well plate (triplicates per time point).
3. Include blank controls-FBS

• ​​Incubation​​: Seal the plate to prevent evaporation, and incubate at ​​37°C​​ for ​​5 days.
• ​​Fluorescence Measurement​​:

1. At each time point, the culture plate is briefly shaken first to prevent the precipitates from aggregating together.
2. Measure fluorescence (​​excitation 488 nm, emission 507 nm​​).

​​4.Data processing

The fluorescence intensity was processed using GraphPad 9.5, and the half-life of the sample was predicted using the Exponential decay - one phase model.

Model: Y = (Y0 – Plateau) * exp(-K*X) + Plateau

Y0 is the Y value when X (time) is zero. It is expressed in the same units as Y,

Plateau is the Y value at infinite times, expressed in the same units as Y.

K is the rate constant, expressed in reciprocal of the X axis time units. If X is in minutes, then K is expressed in inverse minutes.

Tau is the time constant, expressed in the same units as the X axis. It is computed as the reciprocal of K.

Half-life is in the time units of the X axis. It is computed as ln (2)/K.

Span is the difference between Y0 and Plateau, expressed in the same units as your Y values.

To evaluate whether fusion with the self-assembling peptide EFK8 prolongs the half-life of hypoallergenic fusion proteins, we employed EGFP as a model protein incubated in fetal bovine serum at 37 °C for 5 days to simulate in vitro conditions. Fitting the fluorescence decay data to a one-phase exponential model revealed that EFK8-EGFP exhibited a significantly extended half-life (1.820 days) compared to EGFP alone (0.9819 days), nearly doubling its stability (Figure 7). This enhancement is likely attributable to EFK8-mediated oligomerization, which increases molecular size and reduces degradation rate.

Figure 7 Half-life of EGFP and EFK8-EGFP.

The observed half-life of EFK8-EGFP nearly doubled (1.820 days) compared to EGFP (0.9819 days), highlighting the effectiveness of EFK8-mediated oligomerization in enhancing protein stability. The EFK8 peptide may promote the formation of such complexes through hydrophobic and electrostatic interactions, which has been confirmed in the study of viral fusion proteins (Figueira et al., 2017). In this study, a similar strategy could be used to improve in vivo stability. These results support the application of EFK8 fusion proteins in allergen immunotherapy (AIT) for the design of low-allergenic proteins. In this application, prolonging their circulation time in the body can enhance immune exposure and induce immune tolerance, while reducing the frequency of administration.

Developing effective hypoallergenic therapies requires a deep understanding of how structural modifications affect the IgE binding affinity and cytotoxicity. In this part, we employed computational structural biology methods to investigate the mechanisms underlying the reduction in allergenicity of two fusion proteins (EFK8-mPhl p1-GS-mBet v1 and EFK8-mDer p1-GS-mDer p2). The preliminary experimental data indicated that the self-assembly mediated by EFK8 significantly reduced IgE binding and cytotoxicity, which might be attributed to the masking of epitopes or conformational changes due to oligomerization. To validate this hypothesis, we used AlphaFold3 for high-precision prediction of the monomeric and oligomeric structures of the fusion proteins, and then used HADDOCK 2.4 for protein-protein docking simulations to analyze the potential interactions between the fusion constructs and IgE antibodies.

This integrated computational approach allowed us to systematically evaluate how EFK8-mediated oligomerization alters the structural presentation of allergenic epitopes and modulates IgE binding affinity at atomic resolution. The computational predictions were cross-validated with experimental data to establish structure-function relationships critical for rational protein design.

First, AlphaFold3 was utilized to predict the three-dimensional structures of both monomeric and oligomeric forms of our engineered fusion proteins. It generates highly accurate structural models by analyzing amino acid sequences and evolutionary patterns. The model outputs include predicted local distance difference test (pLDDT) scores for each residue, providing confidence metrics for structural reliability. Subsequently, HADDOCK 2.4 was used for protein-protein docking between predicted structures and IgE antibodies. The three-stage docking process includes: (1) rigid-body sampling of orientations, (2) semi-flexible interface refinement, and (3) solvent-optimized molecular dynamics. Incorporating known epitopes and physicochemical parameters (electrostatic/van der Waals forces), HADDOCK generates biologically relevant binding models. Resulting complexes were ranked by HADDOCK scores, which integrate electrostatic, desolvation and van der Waals energy terms.

This integrated computational approach allowed us to systematically evaluate how EFK8-mediated oligomerization alters the structural presentation of allergenic epitopes and modulates IgE binding affinity at atomic resolution. The computational predictions were cross-validated with experimental data to establish structure-function relationships critical for rational protein design.

1. Protein Structure Prediction(AlphaFold3)

(1) Input Amino acid sequences: Group 1 (mPhl p1, mBet v1, and EFK8-mPhl p1-GS-mBet v1); Group 2 (mDer p1, mDer p2, and EFK8-mDer p1-GS-mDer p2)

(2) Output: High-confidence 3D models with pLDDT scores for structural reliability assessment.

2. Protein-Protein Docking (HADDOCK 2.4)

(1) Input: Predicted structure of the fusion protein

(2) Docking Process: A. Rigid-body docking to sample possible binding orientations. B. Semi-flexible refinement of interface residues. C. Explicit solvent refinement for energy minimization.

(3) Key Analyses: A. Cluster selection: Lowest HADDOCK score complex (integrating van der Waals, electrostatic, and desolvation energies). B.Interface analysis: Buried surface area (BSA), hydrogen bonds, and hydrophobic interactions. C.Epitope accessibility: Solvent-accessible surface area (SASA) changes to assess steric hindrance.

3. Validation & Data Interpretation

(1) Energy decomposition to identify key interaction forces.

(2) Z-score evaluation to determine statistical significance of docking poses.

(3) Cross-validation with experimental data to confirm computational predictions.

Molecular docking analysis of this complex revealed a low HADDOCK score (-100.0 ± 5.6) and a significant electrostatic potential contribution (-191.4 ± 22.8), indicating that the assembly process is highly dependent on charge complementarity. This aligns with the Alphafold3 prediction concluding that "electrostatic complementarity dominates assembly." Additionally, the large buried surface area (2176.1 ± 26.7 Ų) and van der Waals energy (-57.2 ± 2.5) further indicate a tightly bound and stable dimer interface. More importantly, this assembly directly leads to spatial shielding of the N-terminal β-sheet (CFEIKCT) in mPhl p1, reducing exposed hydrophilic residues and mechanistically explaining the decreased IgE affinity. Furthermore, the high constraint violation energy (126.3 ± 28.0) suggests potential conformational tension in the linker region, providing direction for subsequent optimizations in linker design. Integrating the diagrams with Alphafold3 predictions reveals that EFK8-guided oligomerization (particularly during higher-order polymer formation) significantly reduces IgE binding capacity through steric hindrance and epitope masking, while electrostatic complementarity serves as the key energy driver for this assembly process. This discovery provides crucial structural biology insights for designing hypoallergenic allergen vaccines. (Figure 8).

Figure 8 (A) Protein structure, (B) dock surface and (C) HADDOCK analysis result of EFK8-mPhl p1-GS-mBet v1 dimer.

Alphafold3 structural prediction indicates that the introduction of the (GS)₄ linker causes the N-terminal α-helix of Der p1 (CWAFSGVAA) to come into excessive spatial proximity with the central β-sheet of Der p2 (KIEIKASI), forming a narrow region of only 4.9 Å. This may hinder antibody binding to key epitopes through steric hindrance effects. Further molecular docking analysis of the EFK8-mDer p1-GS-mDer p2 system via HADDOCK reveals a low HADDOCK score (-110.9 ± 2.2) with stable interfacial binding (van der Waals energy: -74.2 ± 4.3; electrostatic potential: -65.4 ± 10.5), and a buried surface area of 2163.1 Ų, indicating extensive coverage of surface residues (Figure 9). Should this system form higher-order multimers, the steric hindrance between adjacent subunits is expected to further obscure the IgE epitopes on mDer p1 and mDer p2, providing a structural explanation for the reduced immunogenicity of the fusion protein.

Figure 9 (A) Protein structure, (B) dock surface and (C) HADDOCK analysis result of EFK8-mDer p1-GS-mDer p2 dimer.

Through computational simulations, we revealed that EFK8-mediated fusion protein oligomerization may physically shield sensitizing epitopes, thereby reducing IgE binding capacity. However, the current model still has the following limitations and areas for improvement:

(1)Oligomer Quantification and Morphology Validation: Determine actual oligomer size and distribution via techniques like Dynamic Light Scattering (DLS) to provide more accurate oligomeric state constraints for modeling.

(2)Flexible Linker Replacement: Test more flexible linkers (e.g., (GGGGS)n) or rigid linkers (e.g., α-helix-forming peptides) to balance domain independence and interactions.