Abstract

This project aims to develop a multi-target eyedrop that can tackle glaucoma. We have iteratively engineered our strategies over the past months to develop two fusion proteins, FT and BC, aimed at addressing the two primary causes of open-angle glaucoma: high intraocular pressure and retinal ganglion cell apoptosis. By using the engineering cycles, we will describe our designing, building, testing, and learning phases. In the next section, we will explain how we applied the principles of engineering design thinking to successfully finish our project.

Cycle 1

Design 1.1

In this first cycle, our primary objective was to establish a foundational design for a fusion protein utilizing PEP-1 and FWT, which consists of FRATtide, Wnt3α, and TP1. Additionally, we incorporated another fusion peptide known as BC, which consists of BDNF and CDR1, designed to help prevent apoptosis and increase retinal ganglion cells’ survival rates. We constructed a plasmid for the respective fusion peptides, focusing on how each element contributes to the overall functionality of the fusion protein.

We also decided to incorporate the recombinant plasmid pUC18 into our strategy primarily for gene cloning and amplification. pUC18 is well-suited for this purpose due to its small size and high copy number, which enables efficient replication in E. coli DH5-α cells. [1]

For gene and protein expression, we utilized the DH5-α and BL21 strains. DH5-α was selected for its high transformation efficiency and stable plasmid maintenance [2], making it ideal for cloning and amplifying plasmid DNA. This strain is particularly effective for introducing recombinant DNA constructs, including fusion peptides, into cells for plasmid amplification. Moreover, BL21 was chosen for its capability of high-level protein expression, especially in the production of recombinant proteins. The T7 RNA polymerase gene in BL21 facilitates strong transcription of T7 promoter-driven target genes, allowing us to effectively express our fusion peptide constructs and obtain the desired peptides. [3]

Fig 1 FWT Benchling plasmid design (pUC18).

Fig 2 BC Benchling plasmid design (pUC18).

Fig 3 FWT flow

Fig 4 BC flow

The design process involved careful consideration of the roles of each component. In FWT, FRATtide plays a crucial role in inhibiting GSK3, thereby contributing to the higher expression of Axin2 and MMPs. Both of them are responsible for degrading the ECM resistance of aqueous humor. Wnt3α is one of the members of the Wnt signal family, which can benefit from the Wnt/β-catenin signaling pathway functions. TP1, a peptide extracted from ginsentide, was included for its known properties in enhancing NO production and muscle relaxation. In BC, BDNF activates PI3K, MAPK, and PLCy pathways, which can prevent cell apoptosis; and also lower the oxidative stress and eventually facilitate neuroprotection. On the other hand, CDR1 can inhibit caspase-3 indirectly to minimize the RGC death rate.

Build 1.2

Fig 5 Alphafold design for Wnt3α.

Fig 6 Alphafold position error graph of Wnt3α.

Following the design phase, we moved into the lab implementation. We constructed the gene and transformed it into DH5-α and E. coli for their respective functions. Both the transformation of BC and FWT succeeded.

Fig 7 Culture of transformed DH5-α.

Fig 8 Culture of transformed BL21.

We will then perform plasmid purification and protein purification to obtain high-quality, purified plasmid and protein. Initial results from both gel electrophoresis and SDS-PAGE suggested that our fusion protein was expressed successfully, leading us to believe that our design was functioning as intended.

Fig 9 FWT Gel electrophoresis.

Fig 10 BC Gel electrophoresis.

Fig 11 SDS-PAGE result of FWT and BC.

Fig 12 Protein purification.

TESTING 1.3

Subsequent testing revealed significant limitations in the performance of the evaluated components. An ELISA assay was performed to assess binding affinity. Results from the ELISA are generally interpreted based on two key criteria: visual color intensity and the quality of the standard curve. Ideally, the intensity of yellow color developed in the assay should be directly proportional to the amount of target protein in the sample, indicating a successful enzymatic reaction. Furthermore, the standard curve must exhibit a high degree of fit to the data points, which is quantified by the coefficient of determination (R²). An R² value of 0.99 is generally expected for reliable quantification; this shows that the assay measures target analyte concentrations reliably over the tested range. A lower R² value would suggest subpar assay performance, necessitating troubleshooting and a follow-up round of testing.

In our initial ELISA testing, an ideal standard curve quality (R²) was obtained, confirming the efficacy of its design and functionality. The presence of robust protein for the BC component was demonstrated, as evidenced by a strong yellow signal, providing a solid foundation for further development of this component. In contrast, the FWT component showed no detectable protein presence, indicated by the absence of a yellow signal and lack of absorption.

LEARN 1

As a response to these findings, we decided to modify our approach. We hypothesized that the presence of Wnt3α was detrimental to the protein's ability to fold correctly and bind to its intended receptors.

Additionally, we hypothesized the need for a more effective E. Coli strain for more protein expression. Consequently, we removed Wnt3α from our construct and replaced it with PEP-1 FT, and we replaced BL21 with SHuffle cell, which we anticipated would enhance both the folding and binding potential of the fusion protein cycles, and increase the amount of proteins expressed, respectively.

Finally, the recombinant plasmid pUC18 proved to have a better cloning efficiency, but the protein yields from the results were insufficient for subsequent experiments. This signals a need for a different recombinant plasmid, designed to increase protein expression.

Cycle 2

Design 2.1

In the second cycle, we started by redesigning our fusion peptide FWT by removing Wnt3α, leading to a modified fusion peptide now referred to as FT. We conducted experiments with BC without further modifications, as it yielded positive results in all tests during cycle 1. Additionally, while BL21 could express our protein, the yield was insufficient. Therefore, we replaced BL21 with the more effective SHuffle cell for improved expression.

Additionally, we designed a new recombinant plasmid, pET-IDT, dedicated to enhancing protein expression levels. It excelled in expression due to its strong IPTG-inducible T7 promoter and lac operator system. This vector, when paired with specialized host strains like E. coli SHuffle cells, supported enhanced protein folding and post-translational modifications crucial for our eyedrop. [4]

Fig 13 FT Benchling plasmid design (pet-IDT).

Fig 14 BC Benchling plasmid design (pet-IDT).

Fig 15 FT flow

Fig 16 BC flow

Build 2.2

We designed SHuffle cells to avoid periplasmic export or refolding from inclusion bodies by directly generating active, disulfide-bonded proteins (such as antibodies, enzymes, or cytokines) in the cytoplasm. Compared to common strains like BL21, SHuffle cells offer improved folding accuracy for proteins with multiple disulfide links, leading to enhanced activity and solubility. In SHuffle cells, they express disulfide bond isomerase DsbC, which provides an oxidizing cytoplasmic environment that facilitates the production and repair of disulfide bonds within the cytoplasm, unlike BL21.

The synthesis of properly folded and functional proteins—including those that are frequently inactive or insoluble when expressed in BL21—is made possible by this environment. Furthermore, SHuffle provides multiple alternatives for gene expression by supporting T7 promoters. For disulfide-bonded proteins, the yields from SHuffle cells are frequently on par with or superior to those from conventional periplasmic expression systems. This makes SHuffle particularly advantageous for expressing complex fusion peptides, such as those in your glaucoma eyedrop project, as it ensures the production of these peptides with correct structural conformation, increased solubility, and higher therapeutic activity. [5]

To enhance the delivery and therapeutic efficacy of our fusion peptide drugs within the eye, we utilized nanocarrier liposomes as advanced delivery vehicles. These liposomes facilitate improved penetration through ocular barriers, ensuring that the eyedrop reaches deeper target tissues with higher efficiency. This targeted delivery system also helps reduce systemic exposure and off-target effects, thereby minimizing potential side effects and increasing the safety profile of the treatments. Specifically, for the FT drug, we engineered the liposomes to carry and direct the peptides toward pathways involving CTGF and LRP5/6, which play key roles in ocular health and disease modulation. For the BC drug, liposomes target the NGF-TrkA signaling axis, enhancing selective receptor binding and downstream therapeutic effects. Upon reaching the target cells, these nanocarrier liposomes interact with specific receptors on the cell surface. This interaction triggers receptor-mediated endocytosis, a process in which the cell membrane engulfs the liposome-drug complex, internalizing it into the cell. This mechanism ensures efficient intracellular delivery of the drug, allowing it to reach its site of action within the cell. By leveraging these tailored liposomal delivery systems and receptor-mediated uptake, we achieve more precise drug localization, sustained release, and improved bioavailability, all of which contribute to maximizing clinical benefits while maintaining safety in treating eye conditions. [6][7][8]

Specifically, for the FT drug, we engineered liposomes to carry and direct the peptides toward pathways involving CTGF and LRP5/6, which play key roles in ocular health and disease modulation.

Finally, for the BC drug, liposomes target the NGF-TrkA signaling axis, enhancing selective receptor binding and downstream therapeutic effects. By leveraging these tailored liposomal delivery systems, we achieve more precise drug localization, sustained release, and improved bioavailability, all of which contribute to maximizing the clinical benefits while maintaining safety in the treatment of eye conditions.

We will conduct the Cycle 2 experiments in the same manner as Cycle 1, employing SDS, gel electrophoresis, and ELISA to rigorously assess our protein. Once we've addressed all potential improvements in our experiments, we'll proceed to cell line engineering.

We conducted gel electrophoresis, and the results indicate that our DNA expression in bacterial cells for the newly designed plasmid was successful.

Fig 17 Culture of transformed DH5-α.

Fig 18 Culture of SHuffle cells.

As round 1, the E. coli containing our recombinant plasmids were cultured, lysed and underwent protein purification.

Fig 19 BC and FT Gel electrophoresis.

Fig 20 SDS-PAGE result of FT and BC.

Repeating the experiments from the initial cycle, we examined the presence of our recombinant plasmid using SDS-PAGE and gel electrophoresis. The results from SDS-PAGE confirmed the presence of our protein once again, and both transformations for BC and FT were successful. This success allows us to move forward with additional testing, which includes conducting ELISA kits. Furthermore, we observed a higher level of protein expression in the culture of SHuffle cells compared to that of BL21, which supports our hypothesis.

Testing 2.3

In the BCA test, we will test the protein concentration in our drugs.

Two BCA assays were performed: one before protein refolding and one after protein refolding. Both sets of results were within the expected range. From Figure 21, it can be concluded that the proteins possessed a high concentration prior to refolding. Figure 22 indicates the optimal pH condition under which the proteins refolded most effectively, yielding the highest concentration. The purple coloration observed in the wells corresponds to protein concentration, as the intensity of this purple color, measured by absorbance at 562 nm, is directly proportional to the amount of protein present in the sample. The observed gradual change in color intensity across the dilution series confirms that the results meet the expected trend.

Fig 21 BCA RESULTS BEFORE PROTEIN REFOLDING

Fig 22 BCA RESULTs AFTER PROTEIN REFOLDING

In ELISA tests, we will again test the binding affinity of the fusion peptides.

The results revealed strong protein presence in BC as seen visually by the yellow present in the result pictures; on top of that, the standard curve quality is optimal, for PI3K, it has a R² value of 0.997, for ANP32A, it has a R² value of 0.998, for TrkB, it has a R² value of 0.9967, further validating the usage of BC.

The results for FT followed suit, first with intense yellow colors in its results, paired with the desirable standard curve quality as well, for PI3K, it has a R² value of 0.997, for NOS, it has a R² value of 0.9991, for GSK3, it has a R² value of 0.9972, which means FT had successful results for all three experiments.

We have conducted absorbance tests using the ELISA kit assays, and the results are presented below.

Fig 23 ELISA results of ANP32A

Fig 24 ELISA graph of ANP32A

Fig 25 ELISA results of TrkB, PI3k, NOS, GSK3.

Fig 26 ELISA graph of TrkB.

Fig 27 ELISA graph of PI3K.

Fig 28 ELISA graph of NOS.

Fig 29 ELISA graph of GSK3.

After confirming the binding affinity of our peptide via ELISA, the next step is to perform functional testing on actual cell lines. We plan to evaluate the therapeutic effects of the peptide on mouse retinal ganglion cells and human trabecular meshwork cells (HTMC). This will determine whether the peptide exerts its therapeutic potential in relevant human cellular models. We will apply our fusion peptide BC to mouse retinal ganglion cells (RGCs) and assess apoptosis rates using the Annexin V-FITC/PI Apoptosis Detection Kit. Additionally, FT will be administered to human trabecular meshwork cells (HTMCs) to evaluate nitric oxide (NO) production, measured by the fluorescent probe DAF-FM DA.

Learn 2

The successful results from several different tests confirm our hypothesis that our previous fusion peptide, FWT, was negatively affected by Wnt3α.

In addition to the successful results for our fusion peptides, the fact that the optimized culture yields a significantly higher amount of protein than the BL21 culture we performed in cycle 1 shows how the structure of SHuffle cells affects the rate of expression and, consequently, the protein's ultimate yield.

Additionally, in Cycle 1, pET-IDT serves as an ideal replacement for pUC18 because it enables the reliable synthesis of therapeutic proteins that are both biologically active and correctly folded. Several of our peptides require the production of disulfide bonds for action, and the oxidative cytoplasmic environment provided by E. coli SHuffle strains improves this process.

Through these two rounds of experiments, we have discovered that plasmids offer distinct advantages based on the vector and host system employed. Plasmids are natural extrachromosomal DNA elements found in bacterial cells, providing genetic advantages such as antibiotic resistance. In our project, we initially used the pUC18 plasmid in conjunction with the E. coli strain DH5-α for cloning, while utilizing BL21 cells for protein expression. In Cycle 2, we replaced pUC18 with the pET-IDT plasmid for protein expression.

pUC18

Small plasmid size (2.7 kb) allows easy manipulation and cloning of large DNA fragments.

High copy number in E. coli DH5-α enables robust plasmid replication and gene amplification.

pET-IDT

Strong IPTG-inducible T7 promoter enables tightly regulated, high-level expression of therapeutic proteins in specialized strains like E. coli SHuffle.

Lac operator allows precise transcriptional control, minimizing unwanted basal expression of potentially toxic proteins.

Compatible with host strains promoting disulfide bond formation and proper protein folding, critical for biologically active glaucoma therapeutics.

Ultimately, these results further demonstrate that our approach of constructing fusion peptides as a solution for glaucoma is both feasible and achievable.

Conclusion

Throughout the two cycles of our engineering project, we have gained valuable insights into the challenges we faced and the complexities of peptide modification.

After modifying the fusion peptide by removing one of the components from our initial fusion protein’s design, Wnt3α, and replacing BL21 with SHuffle cells for protein synthesis, we successfully increased the stability of our peptide and the amount of proteins expressed. The function of our peptide has also been modified. The successful lab results, including the ELISA results, indicate the improvement of our peptide design.

In cycle 2, we switched to pET-IDT, upon realizing the deficiencies of pUC18, which offers strong, IPTG-inducible T7 promoter-driven expression and works well in DH5-α for cloning and Shuffle cells for high-yield protein production. This system ensures robust, tightly controlled expression and proper protein folding, optimizing production of active therapeutic proteins for our glaucoma treatment.

These modifications deepened our insight into peptide dynamics and highlighted the importance of flexibility in experimental design. Throughout the project, we faced challenges that sharpened our critical thinking and problem-solving abilities, teaching us to overcome obstacles with innovative solutions. This ongoing process not only advanced our technical skills but also reinforced the vital roles of resilience and teamwork in scientific research. Together, these experiences strengthened both our scientific understanding and collaborative approach, laying a strong foundation for future work.

Reference

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