The Problem


Celiac disease is an autoimmune condition in which the body’s immune system is triggered by the presence of gluten. Over time, this immune response damages the small intestine, impairing its ability to absorb nutrients. The resulting symptoms are often painful, harmful, and significantly impact the quality of life of those affected [1]. Gluten is a protein found in wheat, barley, and rye [2]. Specifically, it is the gliadin subunit of gluten that provokes the immune reaction [3].

Gliadin subunit protein structure
Figure 1. 3D structure of Alpha/beta-gliadin A-III from Triticum aestivum (wheat), from Alpha Fold[4].
An estimated 1 in 114 Canadians (about 1%), are affected by celiac disease [1]. Alarmingly, it is believed that up to 85% of cases remain undiagnosed [5], underscoring both the difficulty of obtaining a diagnosis and the wide variation in symptoms and sensitivity to gluten from person to person. Currently, the only way to manage Celiac Disease is through strict, lifelong adherence to a gluten-free diet [6]. Even trace amounts of gluten can trigger a reaction, making the risk of cross-contamination a constant concern. Everyday activities, such as dining out with friends becomes stressful and potentially hazardous.


highlighted toast among the 114 toasts
Figure 2. Illustration of how celiac disease affects approximately 1 in 114 Canadians, represented by the highlighted toast among the crowd.

The Goal


Our goal is to address the risk of cross-contamination by engineering a safe probiotic that both degrades gluten (Aim 1) and blocks its recognition by the immune system (Aim 2).

  • Aim 1: The probiotic secretes enzymes that cleave gliadin subunits into fragments too small to be detected by the immune system.
  • Aim 2: The probiotic produces peptide “caps” that bind remaining gliadin fragments, masking immune recognition sites and preventing uncleaved gluten from causing a reaction.

Our Approach


To achieve our goal, we need an enzyme capable of targeting gluten or gluten-like proteins. Gluten is rich in the amino acids proline and glutamine [2], so we focused on finding an enzyme with specificity toward these residues. Our research led us to Aspergillus niger prolyl endopeptidase (AN-PEP), a well-characterized enzyme that cleaves at proline residues and is already used in the brewing industry to reduce gluten [7]. Notably, AN-PEP was used by UT Austin in their 2024 iGEM project, where it was identified as a promising tool for gluten degradation [8]. However, their team faced difficulties cloning the enzyme, the same major obstacle we encountered in our own work. While we were unable to fully overcome this cloning challenge, our progress reinforces the importance of developing strategies to reliably express AN-PEP and highlights it as a key step toward realizing an effective probiotic therapy.

Schematic representation of the bonds cleaved by prolyl endopeptidase (PEP)
Figure 3. Schematic representation of the bonds cleaved by prolyl endopeptidase (PEP), from [9].

In parallel with enzyme degradation, our design also incorporates peptide caps that bind to the gliadin fragments and mask immunogenic epitopes. Specifically, we focused on the 33-mer gliadin peptide, one of the most immunotoxic fragments of gluten due to its dense concentration of T-cell epitopes [10].

To block recognition of this region, we researched short peptides capable of binding directly to gliadin. Our work led us to two protein caps, p61 and p64, both of which have demonstrated strong affinity for gliadin and the potential to shield its epitope-rich regions [11]. These peptide “caps” provide a second layer of protection, designed to reduce the likelihood of immune system activation even in the presence of uncleaved gluten fragments.

For more details on experiments, check out our wet lab section!

Future Steps


Our project set out to tackle one of the most persistent challenges for people with celiac disease: accidental gluten exposure through cross-contamination. While our design demonstrates a strong proof of concept, we recognize that it is an early step. Future work could refine these mechanisms and expand their effectiveness toward handling cross-contamination. Beyond the technical aspect, our project also aims to raise awareness about the daily struggles faced by individuals with celiac disease and highlight the importance of safe gluten-free options.


References


  1. Celiac Disease. (n.d.). Canadian Digestive Health Foundation. Retrieved October 1, 2025, from https://cdhf.ca/en/digestive-conditions/celiac-disease/
  2. What is gluten? - Dr. Schär Institute. (n.d.). Retrieved October 1, 2025, from https://www.drschaer.com/uk/institute/a/definition-gluten
  3. Barone, M. V., Troncone, R., & Auricchio, S. (2014). Gliadin Peptides as Triggers of the Proliferative and Stress/Innate Immune Response of the Celiac Small Intestinal Mucosa. International Journal of Molecular Sciences, 15(11), 20518–20537. https://doi.org/10.3390/ijms151120518
  4. AlphaFold Protein Structure Database. (2025). AlphaFold Protein Structure Database. Ebi.ac.uk. https://alphafold.ebi.ac.uk/entry/P04723
  5. Gluten Intolerance Remains Largely Undiagnosed in Canada. (n.d.). Retrieved October 1, 2025, from https://temertymedicine.utoronto.ca/news/gluten-intolerance-remains-largely-undiagnosed-canada
  6. Future Therapies. (n.d.). Celiac Disease Foundation. Retrieved October 1, 2025, from https://celiac.org/about-celiac-disease/future-therapies-for-celiac-disease/
  7. Randomized clinical trial: Effective gluten degradation by Aspergillus niger-derived enzyme in a complex meal setting | Scientific Reports. (n.d.). Retrieved October 1, 2025, from https://www.nature.com/articles/s41598-017-13587-7
  8. UT Austin iGEM 2024. (2024). Igem.wiki. https://2024.igem.wiki/austin-utexas/results
  9. Scherf, K. A., Wieser, H., & Koehler, P. (2018). Novel approaches for enzymatic gluten degradation to create high-quality gluten-free products. Food Research International, 110, 62–72. https://doi.org/10.1016/j.foodres.2016.11.021
  10. Structural Basis for Gluten Intolerance in Celiac Sprue | Science. (n.d.). Retrieved October 1, 2025, from https://www.science.org/doi/10.1126/science.1074129
  11. Chen, T., Hoffmann, K., Östman, S., Sandberg, A.-S., & Olsson, O. (2011). Identification of gliadin-binding peptides by phage display. BMC Biotechnology, 11, 16. https://doi.org/10.1186/1472-6750-11-16