In this cycle, we designed and produced recombinant hypoallergenic derivatives derived from six common allergens: Phl p1 and Bet v1 (pollen allergens), Der p1 and Der p2 (dust mite allergens), and Met e1 and Cyp c1 (seafood allergens). Through rational protein design, we eliminated or disrupted key conformational IgE-binding epitopes while strategically preserving T-cell epitopes, and we have named these hypoallergenic derivatives as: mPhl p1, mBet v1, mDer p1, mDer p2, mMet e1, mCyp c1 (Table 1). This approach maintains the immunogenicity required for inducing immune tolerance while significantly reducing the risk of triggering allergic reactions.

Table 1 Design Principles for Hypoallergenic protein Sequences

The plasmids were designed using the pET-32a vector, which features a T7 promoter for high-level expression, a His-tag for simplified purification, and a Trx tag to enhance soluble expression of the target allergen protein. The gene fragments encoding the engineered ​​hypoallergenic proteins​​ were first amplified via ​​PCR​​. The pET-32a vector was then digested with the corresponding ​​restriction enzymes​​ (BamHI and XhoI). Following purification, the target gene fragments and vector were assembled using ​​homologous recombination​​ (Figure 2).

Figure 2 Schematic diagram of the plasmid construction.

​ As shown in Figure 3, PCR products and linearized vector were validated via agarose gel electrophoresis. Target gene fragments (330-720 bp) were confirmed for purity and accuracy through staining and UV visualization. Following gel extraction and purification, six allergen genes (mPhl p1, mBet v1, mDer p1, mDer p2, mCyp c1, mMet e1) were inserted into the linearized pET32a vector using homologous recombination technology, completing recombinant plasmid construction.

Figure 3 Agarose gel electrophoresis results of (A) gene amplification and (B) vector linearization.

The recombinant products were then transformed into E. coli Top 10 competent cells, and positive clones were selected on LB agar plates, respectively. Colony PCR and DNA sequencing verified the correct insertion and integrity of the allergen genes. Selected positive clones were sequenced, and the sequence data confirmed that each plasmid construct contained the correct allergen gene sequence with high fidelity (Figure 4). The validated plasmids were subsequently transformed into E. coli Origami (DE3) competent cells to enhance soluble expression of the recombinant hypoallergenic proteins.

Figure 4 (A) Transformation, (B) colony PCR and (C) Sequencing results of plasmids.

To validate whether our engineered hypoallergenic derivatives indeed exhibit reduced allergenicity (IgE binding capacity), we also produced the corresponding native allergens. Notably, since native rDer p1 requires glycosylation and cannot be produced in E. coli, it was commercially purchased from WOLCAVI BIOTECH. For the other five native allergens (rPhl p1, rBet v1, rDer p2, rMet e1, rCyp c1), we introduced reversion mutations via primers, obtained the fragments through PCR, and subsequently constructed the recombinant plasmids using homologous recombination (Figure 5).

Figure 5 Schematic diagram of the plasmid construction of native allergens.

As shown in Figure 6, PCR products were validated via agarose gel electrophoresis. Target gene fragments were confirmed for purity and accuracy through staining and UV visualization. Notably, due to multiple mutation sites in rCyp c1, its gene fragment (156 bp) was assembled from synthesized oligos and then fused with rCyp-AMP (2847 bp) via overlap PCR, yielding a 2984 bp product. We synthesized the rPhl p1 and rMet e1 fragments in the company. After PCR and agarose gel electrophoresis, the pET32a vector was observed to have a band near 5000 base pairs. Both rPhl p1 (777 base pairs) and rMet e1 (876 base pairs) were of the expected size.After the fragments were recovered through gel cutting, we used homologous recombination to recombine them.

Figure 6 Agarose gel electrophoresis results of gene amplification.

Next, we transferred the recombinant plasmid into the E. coli Top 10 bacteria, and then screen out the positive clones on the LB culture medium plate. The results of colony PCR and DNA sequencing indicated that we successfully obtained the plasmid with correct sequencing (Figure 7). These verified plasmids were subsequently transferred into E. coli Origami (DE3).

Figure 7 (A) Transformation, (B) colony PCR and (C) Sequencing results of plasmids.

1. Protein expression and purification

Next, we expanded the cultivation and used IPTG to induce the expression of the hypoallergenic protein derivatives and native allergens. Our goal was to express and purify the recombinant allergen proteins for subsequent research. After cell lysis and centrifugation, we performed SDS-PAGE to identify the protein expression and purification conditions. The SDS-PAGE analysis results showed that all six hypoallergenic protein derivatives and five native allergens were successfully expressed, and the molecular weights were consistent with the expectations (Figure 8A).

Among them, mPhl p1, mBet v1, and mDer p1 were mainly expressed in the precipitate part, with less background proteins and high purity. These insoluble proteins were then denatured and refolded to obtain soluble proteins. For the three soluble-expressed proteins: mDer p2, mMet e1, and mCyp c1, and the five natural allergen proteins (expressed in both the supernatant and the precipitate), we used nickel columns to purify them separately. The SDS-PAGE results indicated that we successfully obtained highly pure proteins (Figure 8B-C).

Figure 8 SDS-PAGE results of (A) hypoallergenic protein expression, (B) natural allergen proteins, and (C-D) protein purification.

2. Protein quantitative analysis

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 = 0.9909) with the regression equation Y = 0.001403x + 0.004835 (Figure 9). All absorbance measurements at 595 nm were performed in triplicate, and sample concentrations were interpolated based on the standard curve (Table 2).

Figure 9 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.

3. IgE affinity test of allergen protein

Next, 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 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 10A). 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 10B, 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.

Figure 10 IgE affinity of allergen proteins. (A) IgE standard curve, (B) Comparison of natural allergens and hypoallergenic derivatives.

4. Cell proliferation experiment test

Building upon the IgE-binding affinity results, we further 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.

After incubating cells with each protein at concentrations of 1 nM, 10 nM, and 100 nM for 24 h and 48 h, significant differences in cytotoxicity profiles were observed. For mBet v1 and mCyp c1, at all tested concentrations and time points, there was almost no significant effect on cell viability, demonstrating extremely high biocompatibility (Figure 11B, F). For mPhl p1 and mMet e1, the toxicity showed a clear time and concentration dependence (Figure 11A, E). 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 11C, D).

Figure 11 The results of the cell proliferation experiment.

​​ The experimental results demonstrated that our engineered hypoallergenic derivatives ​​significantly reduce IgE-binding affinity​​, confirming the effectiveness of ​​rational epitope disruption​​ in minimizing allergenicity. This reduction in IgE reactivity aligns with the core goal of enhancing the safety profile of allergen-specific immunotherapy (AIT). Furthermore, ​​CCK-8 cytotoxicity assays​​ revealed that these hypoallergenic variants exhibit minimal adverse effects on cell viability, supporting their biocompatibility and potential for in vivo applications.

To further broaden the therapeutic scope of AIT, we plan to develop ​​fusion constructs​​ by concatenating multiple hypoallergenic units derived from the same allergen category and incorporating ​​self-assembling peptides​​. This design aims to promote the formation of ​​stable oligomers or nanoparticles​​, which are expected to ​​prolong circulatory half-life. Such a strategy could enable synchronized multi-allergen delivery while maintaining a favorable safety profile, ultimately improving treatment efficacy and patient compliance.

To address the issue of multiple allergic reaction sensitivities and simplify the treatment plan, we have designed and manufactured a new type of fusion protein. This protein integrates multiple modified hypoallergenic derivatives that trigger similar immune responses into a single molecular structure. This strategy aims to achieve broad therapeutic effects, potentially avoiding the cumbersome process of multiple separate injections, thereby enhancing treatment efficiency and improving patient compliance. To achieve this, we investigated and selected the EFK8 self-assembling peptide (FEFEFKFK), which undergoes oligomerization driven by hydrophobic stacking of phenylalanine residues and electrostatic interactions of lysine residues. This process forms stable nanostructures that increase molecular size and reduce renal clearance, thereby prolonging the circulation half-life (Song et al., 2023).

Figure 12 The assembly process of EFK8 self-assembling peptide (Ding & Wang, 2017).

We assembled short EFK8 oligonucleotide fragments firstly. Subsequently, we separately amplified single hypoallergenic derivative gene fragments with homologous arms through PCR. Then, we used overlapping extension PCR to splice these fragments together to generate the complete fusion allergen gene sequence. Finally, we performed restriction endonuclease digestion and ligation on the purified fusion gene and linearized vector backbone to obtain the desired recombinant plasmid product. Furthermore, we also constructed a plasmid that integrates the EFK8 peptide and the enhanced green fluorescent protein EGFP, which was used for the subsequent determination of the half-life (Figure 13).

Figure 13 Schematic diagram of the plasmid construction.

As shown in Figure 14, PCR products were validated via agarose gel electrophoresis. Target gene fragments were confirmed for purity and accuracy through staining and UV visualization Figure 10A-B. Then, we used overlapping extension PCR to splice these fragments together to generate the complete fusion allergen gene sequence (Figure 14C). After the fragments were recovered through gel cutting, we used homologous recombination to recombine them.

Figure 14 Agarose gel electrophoresis results of gene amplification.

Next, we transferred the recombinant plasmid into the E. coli Top 10 bacteria, and then screen out the positive clones on the LB culture medium plate. The results of colony PCR and DNA sequencing indicated that we successfully obtained the plasmid with correct sequencing (Figure 15). These verified plasmids were subsequently transferred into E. coli Origami (DE3).

Figure 15 (A) Transformation, (B) colony PCR and (C) Sequencing results of plasmids.

1. Protein expression and purification

We expanded the cultivation scale and used IPTG to induce the expression of the natural allergen proteins. The EFK8-mPhl p1-GS-mBet v1 (FPB) and EFK8-mDer p1-GS-mDer p2 (FDD) proteins are both expressed in the precipitate. Subsequently, a denaturation-reconstitution procedure is required to restore their soluble state. The EFK8-mMet e1-GS-mCyp c1 (FMC) protein was expressed in both the supernatant and the precipitate. We purified the soluble FMC protein using a nickel column. The SDS-PAGE results indicated that we were able to successfully purify this protein (Figure 16).

Figure 16 SDS-PAGE results of (A) protein expression and (B) purification.

2. Protein quantitative analysis

Protein concentrations of purified fusion 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 17). All absorbance measurements at 595 nm were performed in triplicate, and sample concentrations were interpolated based on the standard curve (Table 3).

Figure 17 BSA standard curve test by Bradford assay of
(A) The absorbance values of the BSA standard samples. (B) Fitting results of the standard curve.

Table 3 Protein quantification results.

3. IgE affinity test of allergen protein

As illustrated in Figure 18A, 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 18B). 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.

Figure 18 IgE affinity of allergen proteins. (A) IgE standard curve, (B) Comparison of single and fusion hypoallergenic derivatives.

4. Cell proliferation experiment test

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 19A-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 19D-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 19G-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.

​ The CCK-8 results indicated that these fusion constructs (EFK8-PT-mDer p1-GS-mDer p2, and EFK8-PT-mMet e1-GS-mCyp c1) possessed excellent biological safety characteristics and had a negligible impact on cell viability. This highlighted that the fusion strategy not only can expand the coverage of immunotherapy but also enhance biocompatibility. These findings supported the advancement of the fusion of hypoallergenic derivatives towards further evaluation.

Figure 19 The results of the cell proliferation experiment.

5. Half-life test

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 20). This enhancement is likely attributable to EFK8-mediated oligomerization, which increases molecular size and reduces degradation rate.

Figure 20 Half-life of EGFP and EFK8-EGFP.

In the above experimental results, we successfully constructed and expressed the fused hypoallergenic derivative, and tested its allergenicity (IgE affinity), biocompatibility and half-life. We found the significantly reduced IgE binding affinity and cytotoxicity observed in our fusion allergens (EFK8-PT-mPhl p1-GS-mBet v1, EFK8-PT-mDer p1-GS-mDer p2) compared to their monomeric counterparts. We speculated that may be attributed to several synergistic mechanisms facilitated by the fusion strategy and the self-assembling peptide EFK8.

First, fusion-induced ​​steric hindrance​​ and ​​conformational rearrangement​​ likely occlude key IgE-binding epitopes, thereby directly impairing antibody recognition and cross-linking capacity. Secondly, the ​​oligomerization​​ process triggered by EFK8 will increase the molecular size and alter the surface topological structure through hydrophobic and electrostatic interactions, causing conformational changes or masking at specific sites in these fused hypoallergenic derivatives, thereby further leading to the loss of toxic functions. We have predicted the molecular structures of EFK8-PT-mPhl p1-GS-mBet v1 and EFK8-PT-mDer p1-GS-mDer p2, and conducted molecular oligomerization simulations. We found that after oligomerization, some parts of the structures of these low-allergenic derivatives would be covered. For details, please refer to the Model page.

Collectively, these modifications not only diminish immediate hypersensitivity risks but also mitigate direct cellular toxicity, underscoring the potential of fusion-based designs in developing safer and more effective allergen immunotherapies.