Contribution

Contribution

Our Contribution

This year, our team made significant advances in both the expression of recombinant antigen proteins and the development of stable vaccine carriers. We also openly documented negative results and troubleshooting strategies to help future teams.

1. Application of SUMO protein tag on Antigen protein expression

One of our primary focuses was the application of the SUMO protein tag (BBa_K2933038), inspired by the work of the SHSID-China 2024 iGEM team. The SUMO tag has been reported to enhance the solubility of difficult-to-express proteins, such as the IL-18 binding protein used for IBD therapy.

Plasmid map of pET28a-IL18-SUMO-BPa-Fc
Plasmid map of pET28a-IL18-SUMO-BPa-Fc (source).

By applying this tag to our own antigen proteins derived from the flurval protein, we aimed to improve their soluble expression in E. coli.

Schematic Diagram of Recombinant Ferritin Nanoparticle Immunogen Constructs and Expression Vector
Schematic Diagram of Recombinant Ferritin Nanoparticle Immunogen Constructs and Expression Vector.

Despite following established protocols and optimizing IPTG induction conditions, our SDS-PAGE analyses consistently showed that the antigen proteins remained insoluble. This outcome, although initially disappointing, provided important insights: the effectiveness of the SUMO tag is context-dependent and may not overcome the intrinsic challenges of all protein sequences.

SDS-PAGE analysis of soluble expression of recombinant ferritin-based immunogens
Fig10. SDS-PAGE Analysis of Soluble Expression of Recombinant Ferritin-Based Immunogens. SDS-PAGE analysis of soluble protein expression for three recombinant ferritin nanoparticle immunogens (B-(16)₄-F, H1-(16)₄-F, and H3-H1-B-F) in E. coli under different IPTG induction concentrations. Lanes 1–3: B-(16)₄-F with 0, 0.2, and 0.5 mM IPTG; lanes 4–6: H1-(16)₄-F with 0, 0.2, and 0.5 mM IPTG; lanes 7–10: H3-H1-B-F with 0, 0.2, 0.5, and 1 mM IPTG. The molecular weight markers (Marker) are shown on the left. No significant soluble expression of the target proteins was detected in the supernatant under the tested induction conditions.

Recognizing this limitation, we developed and optimized an in vitro protein folding protocol to recover functional proteins from inclusion bodies, thus ensuring the production of our target immunogens. By sharing these negative results and our troubleshooting strategies on our project wiki, we contribute valuable information to the broader synthetic biology community, helping others to navigate similar challenges more efficiently. See more on our Engineering page.

2. Improve of small particle vaccine carrier

In addition to our work on protein expression, we also sought to improve the performance of vaccine carriers. Building on previous approaches such as the use of bacterial outer membrane vesicles (OMVs) by the SZ-SHD 2022 iGEM team, we selected bacterial ferritin as a nanoparticle carrier for antigen display.

Design of OMV protein producing part
Design of OMV protein producing part (source).

This year, our team chose to use the bacterial Ferritin protein as a carrier. We also mentioned this idea on our part page BBa_K4283012. Ferritin offers several advantages, including the ability to protect antigens from enzymatic degradation and to enhance immune stimulation. We engineered ferritin-based nanoparticles to display conserved influenza HA epitopes and rigorously tested their stability through SDS-PAGE analysis.

Schematic Design of Recombinant Ferritin Nanoparticle Immunogen Displaying Conserved Influenza HA Epitopes
Schematic Design of Recombinant Ferritin Nanoparticle Immunogen Displaying Conserved Influenza HA Epitopes.
SDS-PAGE analysis assessing degradation stability of ferritin-based protein
Fig14. Assessment of Degradation Stability of Ferritin-Based Protein by SDS-PAGE. SDS-PAGE analysis of ferritin-based protein samples after overnight incubation at room temperature. M: protein marker; lanes 1–3: ferritin-based protein samples collected from elution fractions. Target protein bands are still detected in the elution fractions after incubation, although partial degradation is observed. The main protein band remains present, indicating that the ferritin-based protein retains stability under these conditions.

Our results demonstrated that these ferritin-based proteins retained their main structural bands after overnight incubation at room temperature, with only minor degradation observed. This confirmed the robustness and suitability of ferritin nanoparticles as stable vaccine carriers under physiological conditions. By documenting our design and results, we provide a valuable example of how ferritin can be repurposed as a versatile platform in vaccine development, potentially benefiting a wide range of applications beyond influenza.

Beyond these technical achievements, our project underscores the importance of transparency and open sharing in scientific research. By openly reporting our unsuccessful attempts as well as our successes, we contribute to a culture of honesty and continuous improvement in the synthetic biology community.