Results

Overview of gene blocks design for protein expression in E. coli

To anchor sRAGE protein onto the collagen layer of patch and preserve the flexibility for replacing sRAGE with other effective proteins (e.g. anti-microbial peptide), we decide to apply the intein system to fuse the collagen binding domain (CBD) with sRAGE.

Accordingly, the C-terminal of sRAGE protein was fused with Npu-DnaE^N, the N-terminal of split intein, while the N-terminal of CBD was fused with Npu-DnaE^C, the C-terminal of split intein. The peptide sequence at the N-terminal of Npu-DnaE^N and C-terminal of Npu-DnaE^C were respectively modified to RGK (sRAGE-RGK-Npu-DnaE^N) and CWE (Npu-DnaE^C-CWE-CBD) for efficient splicing (Cheriyan, Pedamallu, Tori, & Perler, 2013). The 6xHis tag was added on sRAGE-RGK-Npu-DnaE^N and Npu-DnaE^C-CWE-CBD for protein purification (sRAGE-6xHis-RGK-Npu-DnaE^N, 6xHis-Npu-DnaE^C-CWE-CBD, and Npu-DnaE^C-CWE-6xHis-CBD).

Finally, we fused the EGFP protein to sRAGE-6xHis-RGK-Npu-DnaE^N to monitor the interaction between sRAGEs and AGEs (sRAGE-EGFP-6xHis-RGK-Npu-DnaE^N). The expression of fusion protein was under the control of Lac promoter.

Experimental result

Cloning the designed gene blocks into pET15b
To express the sRAGE-EGFP-6xHis-RGK-(Npu-DnaE^N) (sRAGE-Int^N for short), 6xHis-(Npu-DnaE^C)-CWE-CBD (6xHis-Int^C-CBD for short) and (Npu-DnaE^C)-CWE-6xHis-CBD (Int^C-6xHis-CBD for short) protein in E. coli, we ordered the codon-optimized gene blocks from IDT and performed Gibson assembly to insert the gene blocks into pET15b vector. The sequence was verified by Sanger sequencing before transformation into BL21 for protein expression (Figures 2, 3 and 4).

Cloning the designed gene blocks into pET15b
We then induced the expression of sRAGE-Int^N by 1 mM IPTG. The induced protein was harvested at indicated time points (0 hr and 2 hr) at 37℃ (Figure 5).

The induction of sRAGE-Int^N protein in E. coli at 20 °C
Since the induced sRAGE-Int^N protein located in the inclusion bodies, we then try to enhance the solubility by decreasing the induction temperature to 20 °C. The induced sRAGE-Int^N protein was harvested at indicated time points (0 hr, 2hr, 4hr, and 6 hr). Unfortunately, the expressed sRAGE-Int^N protein was still in the insoluble fraction, suggesting the inclusion body’s location (Figure 6).

Solubilizing sRAGE-Int^N protein from inclusion body by 8M urea
To solubilize the insoluble sRAGE-Int^N protein for subsequent experiments, we applied the 8 M urea extraction buffer to the insoluble fraction, and confirmed the efficient extraction (Figure 7).

The preparation of Int^C-6xHis-CBD and 6xHis-Int^C-CBD proteins.
Similar to sRAGE-Int^N, Int^C-6xHis-CBD and 6xHis-Int^C-CBD protein were both expressed in the insoluble fraction, either at 20 °C or 37 °C (Figures 8-11). Therefore, we applied 8 M urea buffer to solubilize the Int^C-6xHis-CBD and 6xHis-Int^C-CBD proteins from pellet fraction (Figures 12 and 13).

The purification of expressed proteins by His-tag
To purify sRAGE-Int^N, Int^C-6xHis-CBD and 6xHis-Int^C-CBD protein, we performed Ni-IDA resin. We first tested a 1-hour binding, followed by washing with PBS containing 100 mM imidazole to remove nonspecific proteins, and then attempted to elute the target protein with PBS containing 600 mM imidazole (Figures 14-16). The SDS-PAGE analysis of eluted protein and beads suggested the poor elution efficiency.

We next increased the concentration of imidazole in elution buffer to 1 M and 2 M, and the SDS-PAGE analysis result show a slight improvement of elution (Figure 17).

We then reduced elution buffer volume to 200 µl. The SDS-PAGE analysis result suggested the better improvement of elution (lane E1). However, most sRAGE-Int^N protein still bound to the beads (Figure 18).

In addition, we examined whether low-pH elution (0.1M glycine buffer, pH 2.5) works. However, the recovery was still poor, and substantial amounts of protein were retained on the resin (Figure 19).

Similar to sRAGE-Int^N, the elution of Int^C-6xHis-CBD and 6xHis-Int^C-CBD proteins was inefficient by 1M imidazole (Figures 20 and 21) or low-pH elution buffer (Figures 22 and 23).

The innate characteristic of intein affects Ni²⁺ resin mediated purification
After multiple attempts, we suspected that the strong retention was caused by the Npu-DnaE intein domain. To support this hypothesis, we performed the His-Tag purification of protein without intein domain by the same elution buffer, showing that the elution failure was not due to experimental operation or buffer composition but specifically related to the presence of the Npu-DnaE intein.
The SDS-PAGE analysis result revealed a distinct band in the elution fraction 1 (E1) for the EGFP-CBD protein lacking the Npu-DnaE intein, indicating efficient elution (Figure 24).

Similarly, the SDS-PAGE analysis result showed a distinct band in the elution fraction 1 (E1) of the EGFP-4A-C7 protein without the Npu-DnaE intein (from 2023 CCU-iGEM), indicating that the protein was efficiently eluted (Figure 25).

Validating intein activity of sRAGE-Int^N, Int^C-6xHis-CBD, and 6xHis-Int^C-CBD
The 8M urea buffer unfold most extracted protein. However, the previous report suggested that the intein splicing would not be affected (Zettler, Schütz, & Mootz, 2009). Accordingly, we mix cell lysate containing sRAGE-Int^N with lysate containing Int^C-6xHis-CBD or 6xHis-Int^C-CBD. The mixtures were incubated at 37 °C, and aliquots were collected at 0, 0.5, and 1 hour, followed by quenching with SDS.
The SDS-PAGE analysis revealed that, after mixing, a new band appeared at the expected molecular weight position corresponding to either sRAGE-EGFP-6xHis-RGK-CWE-6xHis-CBD (named sRAGE-2His-CBD for short) or sRAGE-EGFP-6xHis-RGK-CWE-CBD (named sRAGE-His-CBD for short). The band was more clearly visible on a 6% PAGE gel. However, the band intensity gradually decreased with longer incubation time, which we speculate was due to protein degradation resulting from the use of unpurified samples (Figures 26 and 27).

Since Coomassie-stained analysis of the sRAGE-Int^N lysate revealed a background protein with a molecular weight similar to the expected splicing product, we further performed a specific Western blot assay to verify the presence of the spliced protein. The results showed that the sRAGE-Int^N and Int^C-6xHis-CBD mixture exhibited a distinct band at the expected molecular weight at 0 h, while the sRAGE-Int^N and 6xHis- Int^C-CBD mixture showed the same result after 0.5 h, indicating that the sRAGE-Int^N and Int^C-6xHis-CBD pairing reacted faster and achieved a higher splicing efficiency than the sRAGE-Int^N and 6xHis- Int^C-CBD pairing (Figure 28).

These results indicated that the intein is functional even in the 8 M urea buffer, suggesting the concept of intein splicing mediated fusion protein is workable. However, the intein splicing products were not significant, likely due to the denaturing conditions of 8M urea. Therefore, we concluded that E. coli expression may not be optimal for producing soluble, properly folded protein with intein, and decided to switch to a mammalian expression system to avoid the need for harsh denaturants and improve protein solubility.

Overview of gene blocks design for protein expression in HEK293T

To solve the problem of inclusion body expression of designed protein in E. coli, we decided to shift the expression system to mammalian cell line HEK293T. Since the RAGE originates from trans-membranous protein, we added signal peptide sequence to sRAGE-EGFP-6xHis-Npu-DnaE^N (sRAGE-Int^N for short) for proper expression in HEK293T. Accordingly, the expressed protein should be harvested from culture medium. Furthermore, we mutated the N-glycosylation sites (N25Q and N81Q) on sRAGE to deplete the N-glycan, which increase the binding affinity of sRAGE to AGEs (mut-sRAGE-Int^N). Together with 6xHis-Npu-DnaE^C-CWE-CBD (6xHis-Int^C-CBD for short), and Npu-DnaE^C-CWE-6xHis-CBD (Int^C-6xHis-CBD for short), the gene block of all four protein were codon-optimized and cloned into pcDNA3.1 by conventional molecular cloning technique.

Experimental result

The unexpected 6xHis-T7 tags in pcDNA3.1
The gene blocks of sRAGE-Int^N and mut-sRAGE-Int^N are first cloned into pcDNA3.1 by restriction enzyme BamHI and EcoRI. However, the Sanger sequencing verification indicated the unexpected 6xHis-T7 tag in front of our designed signal peptide sequence (Figure 30), and further examination showed that this unexpected tag per-exist in the vector, suggesting the wrong vector map sequence.

The cytoplasmic expression of 6xHis-T7-sRAGE-Int^N protein
Since the N-terminal tag would abolish the function of signal peptide and hamper the secretion of expressed protein, we must re-construct the sRAGE-Int^N expressing vector. However, the 6xHis-T7 tag is inflame with sRAGE-Int^N, suggesting that this construct (6xHis-T7-sRAGE-Int^N) could still provide information about the location and folding of sRAGE-Int^N protein. Therefore, we first performed fluorescence microscope observation. The result showed the positive EGFP signals after transfecting pcDNA3.1-6xHis-T7-sRAGE-Int^N (Figure 31). The immunocytochemistry (ICC) staining further showed the cytoplasmic location of sRAGE-Int^N protein in HEK293T (Figure 32). Together, this result suggested the proper folding and cytoplasmic location of 6xHis-T7-sRAGE-Int^N protein in HEK293T.

We then performed the Western Blotting (WB) to examine whether the 6xHis-T7-sRAGE-Int^N protein was secreted into culture medium. The WB result indicated that the 6xHis-T7-sRAGE-Int^N remained in the intracellular soluble fraction and was not secreted into the culture medium (Figure 33).

Constructing the pcDNA3.1-sRAGE-Int^N, pcDNA3.1-mut-sRAGE-Int^N, pcDNA3.1-Int^C-6xHis-CBD, and pcDNA3.1-6xHis-Int^C-CBD
To build the correct construct without T7-tag inside, we re-cloned the gene blocks of sRAGE-Int^N, mut-sRAGE-Int^N, Int^C-6xHis-CBD and 6xHis-Int^C-CBD into pcDNA3.1 using HindIII and EcoRI. The sequence was verified by Sanger sequencing before transfection into HEK293T for protein expression (Figures 34-37).

Expression of sRAGE-Int^N and mut-sRAGE-Int^N in HEK293T
To examine whether the sRAGE-Int^N and mut-sRAGE-Int^N protein could properly expressed in HEK293T, we first applied fluorescence microscope to observe the EGFP signal in transfected cells. The result show positive EGFP signal in either pcDNA3.1-sRAGE-Int^N or pcDNA3.1-mut-sRAGE-Int^N transfected cells, suggesting the successful expression of sRAGE-Int^N and mut-sRAGE-Int^N protein (Figure 38).

Detecting and purifying of sRAGE-Int^N and mut-sRAGE-Int^N in HEK293T
To confirm the sRAGE-Int^N and mut-sRAGE-Int^N secretion in culture medium, we performed Immuno-blotting using an anti-His antibody. The result indicate the presence of sRAGE-Int^N and mut-sRAGE-Int^N protein in both cell culture medium and the cell lysate (Figure 39).

We then performed the protein purification using Ni-IDA resin to capture His-tagged proteins. The result indicated that Non-specific proteins were effectively removed by washing with PBS containing 600 mM imidazole. Both sRAGE–Int^N and mut-sRAGE–Int^N were efficiently retained on the Ni-IDA resin, but could not be eluted and remained bound to the resin (Figures 40 and 41).

Detecting protein expression of Int^C-6xHis-CBD and 6xHis-Int^C-CBD in HEK293T
Similarly, Western blot analysis was conducted to examine the expression of Int^C-6xHis-CBD and 6xHis-Int^C-CBD in HEK293T cells. However, no band was observed at the expected molecular weight of 12.65 kDa (Figure 42), suggesting that transfection or protein expression was unsuccessful. Due to time constraints, successful expression could not be demonstrated in this study. Future work will focus on optimizing experimental conditions, such as adjusting transfection parameters, to enhance protein expression efficiency.

The sRAGE–Int^N expressed in HEK293T cells could not be successfully purified, further confirming that the presence of the Npu-DnaE intein interferes with elution and purification. Once Int^C-6xHis-CBD and 6xHis-Int^C-CBD are successfully expressed, we will proceed with purification experiments to further validate this observation. After completing the purification process, intein-mediated splicing experiments will be conducted to generate additional target fusion proteins and evaluate the overall efficiency of the system.

Overview

To independently assess the binding capability of CBD to the hydrogel matrix without interference from other structural domains, such as sRAGE or intein, we constructed EGFP–CBD fusion protein to employ as a fluorescent reporter probe. This construct served as an intein-independent validation module to confirm the intrinsic collagen-binding functionality of CBD prior to its incorporation into the full sRAGE–CBD fusion system.

Design of the EGFP–CBD construct

To verify the hydrogel-binding capability of CBD in isolation, EGFP was genetically linked to CBD via a flexible GGGSGGEF linker (EGFP-linker-CBD) to preserve the structural integrity of CBD and minimize steric hindrance arising from protein–protein interactions. For E. coli expression, the native 6xHis tag and thrombin cleavage site present in the pET15b vector were retained (6xHis-Thrombin-EGFP-linker-CBD). The 6xHis tag enabled affinity purification, while the thrombin cleavage site allowed post-purification removal of the tag to eliminate potential interference with CBD–hydrogel interactions. In contrast, for HEK293T expression, only a 6xHis tag was incorporated without the addition of a thrombin cleavage site (EGFP-6xHis-linker-CBD) , thereby providing a simplified construct optimized for mammalian protein expression.

Expressing EGFP-CBD protein for hydrogel attachment test

The confirmation of correct insertion of designed gene blocks in pET15b
To express the 6xHis-Thrombin-EGFP-linker-CBD (named EGFP-CBD for short) protein in E. coli, we ordered the codon-optimized gene blocks from IDT and performed Gibson assembly to insert the gene blocks into pET15b vector. The sequence was verified by Sanger sequencing before transformation into BL21 for protein expression (Figure 44).

The expression of EGFP-CBD protein in E. coli
We first induced the expression of EGFP-CBD with 1 mM IPTG at 37 °C and collected samples at different time points (0, 2, 4, and 6 hr) (Figure 45A). However, the expressed protein mainly accumulated in the inclusion bodies. To improve solubility, we lowered the induction temperature to 20 °C and repeated the expression under the same conditions. Despite this adjustment, EGFP-CBD was still found in the insoluble fraction, indicating that it remained localized in inclusion bodies (Figure 45B).

Solubilizing EGFP-CBD protein from inclusion body by 8M urea
To solubilize the insoluble EGFP-CBD protein for subsequent experiments, we applied 8 M urea extraction buffer to the insoluble fraction and confirmed efficient extraction (Figure 46A). However, treatment with 8 M urea caused the EGFP to lose its green fluorescence (Figures 46B and 46C).

Switching the expression system in mammalian cells
To solve the problem of inclusion body expression of designed protein in E. coli, we decided to shift the expression system to mammalian cell line HEK293T. We design the new construct with codon-optimized for mammalian cells and cloned the EGFP-CBD into pcDNA3.1 using HindIII and EcoRI. The sequence was verified by Sanger sequencing before transfection into HEK293T for protein expression (Figure 47).

Then, we transfected the EGFP-CBD into HEK293T and verify the successful transfection and expression result with fluorescence microscope (Figure 48), showing the EGFP signals is right where inside the cell.

Finally, Western blot analysis was performed to verify the size of the expressed protein, confirming the presence of the intact EGFP-CBD fusion in the soluble supernatant of cell lysate (Figure 49).

Due to time constraints, we were unable to present the purification results of EGFP–CBD. In future work, once the hydrogel component is fully developed, we will combine the purified EGFP–CBD with the hydrogel to evaluate the binding capability of the CBD domain to our collagen–chitosan hydrogel system.

After checking the expression of EGFP-CBD, we will perform Ni-IDA resin and apply it to collagen-based hydrogel patches and assess attachment using EGFP fluorescence. This will allow us to evaluate the collagen-binding capability of the CBD domain independently of other protein modules.

Next, the final sRAGE–CBD fusion protein, assembled via intein-mediated ligation, will be tested to confirm effective hydrogel anchoring. The functional performance of the hydrogel-bound fusion protein will then be evaluated using ELISA to determine its ability to capture AGEs. Together, these experiments will provide a stepwise validation of both surface attachment and functional integrity before integration into the full therapeutic system.

In the AGE-Thwart, polyurethane (PU) was selected as the outermost protective membrane due to its ability to provide reliable physical protection, preventing external contamination and mechanical friction while reducing the risk of exogenous infection.

For the inner layer contacting wound, collagen and chitosan were chosen. Collagen mimics the extracellular matrix, promotes angiogenesis and collagen deposition, and accelerates tissue regeneration (Cynthia Ann Fleck . 2010). In addition, collagen serves as a suitable platform for anchoring sRAGE by collagen binding domain. Chitosan, on the other hand, possesses inherent antibacterial properties and excellent exudate absorption capacity (Ijaz Bano . 2017).

Inspired by the work of the 2021 iGEM team MR.SA, the original design of patch consisted of three distinct layers: a polyurethane protective layer, a chitosan absorption layer, and a collagen contact layer.

To optimize the patch, we explored the possibility to reduce the complexity of original design. We found that, in article Effects of Chitosan-Collagen Dressing on Wound Healing in Vitro and in Vivo Assays, dressings composed of both collagen and chitosan demonstrate enhanced wound healing compared to those made of collagen or chitosan alone (Min-Xia Zhang, 2021).

So, we redesigned the patch from a three-layered structure to a two-layered structure, integrating collagen and chitosan into a unified contact layer.

Design:
A uniform hydrogel of collagen and chitosan was prepared using a physical crosslinking approach. Collagen and chitosan were individually dissolved in acetic acid and subsequently mixed at a defined ratio, which was then neutralized to pH 7 by 1M NaOH, followed by freeze-drying to obtain hydrogel (Pablo Sánchez-Cid .2022).

In practice, we found that the crosslink of collagen and chitosan mixture by 1M NaOH neutralization cause a sudden pH change to alkaline state, hampering the hydrogel formation. Using NaOH with low concentration partially solves this problem, but increases the final volume of reaction. Since the small volume of crosslink reaction makes it not easy to be measured by convectional pH meter, we decide to perform an acid-base titration modeling to determine the suitable NaOH concentration for crosslink (Figure 1).

Based on modeling result, the following procedure was carried out. Firstly, 775ul of 1% chitosan was added separately to three 6-cm dishes. Secondly, 73.42ul 1M, 734.2ul 0.1M and 7342ul 0.01M NaOH was added to three dishes to raise pH to 6. Lastly, these two dishes were lyophilized.

Result and conclusion:
The chitosan crosslinked with 0.01 M NaOH showed a uniform and intact sheet. Other concentrations, 0.1M and 1M produced incomplete sheets and the structure of these sheets were not uniform (Figure 2).

To sum up, we selected 0.01M NaOH to is crosslink the chitosan and collagen mixture.

Design:
After determining the NaOH concentration in crosslinking, we first apply the 98.421ul 0.01M NaOH to neutralize and crosslink the 2.5ml 3.1%(mg/ml) collagen and 775ul 1%(g/ml) chitosan mixture (1:1).

Result and conclusion:
After freeze-drying, the hydrogel showed a web-like structure rather than forming an intact sheet (figure 3).

We speculated that the reason for the hydrogel forming a web-like structure is the excess amount of NaOH in neutralizing collagen in HCl solution. The base area of container may also cause the uneven distribution of solution.

Design:
We transferred the contents of the previous experiment from 6 cm dishes to 3.5 cm dishes. In the first step, we added 775ul of 1% chitosan separately to two 3.5-cm dishes. Secondly,73.42ul 1M and 734.2ul 0.1 M NaOH was added to two dishes to crosslink chitosan. Lastly, these two dishes were lyophilized.

Result and conclusion:
The hydrogel prepared with 0.1 M NaOH formed an intact sheet, whereas the hydrogel prepared with 1 M NaOH did not (Figure 4).

The result showed that 0.1M NaOH was able to form a complete hydrogel simply by changing the container.

Design:
Due to the running out of collagen, we only have collagen (Enzo Life Sciences, USA) with low concentration (100ug/ml) for testing. We first lyophilized 15 mL of collagen and resolve into 1.5 ml of acetic acid (1 mg/ml). The collagen solution was then mixed with 0.15ml chitosan solution and stirred for 1 hour, then transferred into 3.5 cm dishes. Second, 1.56315mL of 0.1 M NaOH was added to complete the crosslinking. Finally, the hydrogels were lyophilized.

Result and conclusion:
The resulting hydrogel formed a thin film; however, the edges of the film were slightly damaged (figure 5).

In our opinion, this hydrogel was better than the previous one with collagen and chitosan due to our adjustment in concentration of NaOH and container size. However, the hydrogel didn’t form an intact sheet. We thought this was due to the total content of collagen and chitosan being too low. So, we will try to increase the concentration of collagen solutions in the future.

In future work, we plan to increase collagen concentration to fabricate a 1:1 hydrogel. After successfully producing the 1:1 hydrogel, we will adjust the collagen-to-chitosan ratio and conduct detailed structural and antibacterial evaluations to further optimize the patch composition. Scanning Electron Microscopy (SEM) will be utilized to observe the microstructure and quantify the pore characteristics under different collagen-to-chitosan ratios. These analyses will provide insights into how the matrix architecture affects protein immobilization and wound healing performance.

Furthermore, agar diffusion assays will be performed to systematically evaluate the antibacterial efficacy of the hydrogels against common wound pathogens. By correlating the SEM findings with antibacterial assay results, we aim to determine the optimal material ratio that achieves a balance between protein immobilization, antibacterial activity, and biocompatibility.