Part:BBa_K5466035

Constitutive expression of Nb28-S102D Aga2P

The expression of surface sdAb to capture AFB1 from the environment. Controlled by the constitutive promoter TDH3 (BBa_K124002) to ensure that there is always sdAb on the surface to capture AFB1.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Display of Anti-AFB1 Nb

Displaying the NB28-S102D nanobody (Nb) on the yeast cell wall via Aga2P allows the engineered yeast to maximize its surface area with Nbs ready to capture AFB1. Due to its high sensitivity and binding capacity, this method is highly efficient for capturing AFB1 from the environment. We opted to anchor the nanobody to the yeast surface because our goal is to use this in a probiotic yeast to capture AFB1 in the intestine.

If the VHHs were secreted, they could dilute in the vast intestinal environment. By exposing them on the cell, the yeast retains the proteins on its surface, ensuring they do not disperse or degrade easily in the intestinal environment. We expect to prevent degradation by proteolytic enzymes in the digestive tract, keeping the proteins more protected thanks to robutness of the cell wall. Additionally, secreting proteins could disrupt the balance of the intestinal microbiome or trigger an unwanted immune response. By displaying it, we minimize their impact on the intestinal environment and reduce the risk of adverse effects.

This same approach could be applied to capture other toxins or molecules.

AGA2P

AGA2 is an extracellularly-secreted glycoprotein that constitutes the adhesion subunit of a-agglutinin in a-cells. The N-terminal sequence of the protein harbors a signal peptide (SP) required for protein translocation. Its C-terminal sequence acts as a ligand for alpha-agglutinin, promoting binding and aggregation during mating.

AGA2 remains strongly anchored to the cell wall thanks to AGA1, the anchorage subunit of a-agglutinin. AGA1 stays attached to the cell surface through GPI and strongly binds AGA2 via two disulfide bonds.

AG was added to the C-terminus of the signal peptide based on the discovery by Wang et al. (2005) regarding the importance of specific amino acids for proper peptide cleavage. More details can be found on the design page.

AntiAFB1- Nb28-S102D

We choose a sdAb, specifically a VHH, also known as a nanobody (Nb), to capture AFB1 because it is particularly effective due to its high solubility, exceptional stability, and high affinity. Nbs are significantly smaller than conventional antibodies. Recent studies indicate that Nbs can form concave-shaped binding sites for small molecules, similar to conventional antibodies.

The half-maximal inhibitory concentration (IC50) is a metric used to assess the potency of a substance in inhibiting a biological or biochemical function. It represents the concentration of an inhibitory agent (such as a drug) required to reduce a specific biological process or target by 50% in vitro. This target could be an enzyme, a cell, or a cell receptor. In this case, the lower the IC50, the less AFB1 is required to bind the 50% of the nanobodies. Nb 28-S102D has an IC50 of 1.18 ng/mL.

Protein display

Aga2p can utilize either the N- or C-terminus for surface protein display, and it can also use both termini to display two heterologous proteins as part of one fusion protein. We demonstrate that various proteins can be anchored in this manner while retaining their functional activity. In one instance, Lim et al. (2017) achieved dual expression of a fluorescent protein alongside a ligand, receptor, or antibody fragment. This approach reduces both time and cost, streamlining the determination of equilibrium binding constants compared to conventional yeast surface display methods. Additionally, Lim et al. (2017) demonstrate that dual expression of the bioconjugation enzyme Staphylococcus aureus sortase A and its corresponding peptide substrate, within the same Aga2p construct, allows for the measurement of catalytic activity on a non-natural substrate. This method is simpler and more versatile than previously reported approaches.

Previously applied display strategies involved fusion of AGA2P and its signal peptide to the N-terminus of the protein of interest. Wang et al. (2005) showed increased affinity constants for displayed scFvs when AGA2 signal peptide was attached to the N-terminal and the rest of the protein was fused to the C-terminal end of the scFv.

References

Lim, S., Glasgow, J. E., Interrante, M. F., Storm, E. M., & Cochran, J. R. (2017). Dual display of proteins on the yeast cell surface simplifies quantification of binding interactions and enzymatic bioconjugation reactions. Biotechnology Journal, 12(5). https://doi.org/10.1002/biot.201600696

He, T., Nie, Y., Yan, T., Zhu, J., He, X., Li, Y., Zhang, Q., Tang, X., Hu, R., Yang, Y., & Liu, M. (2021). Enhancing the detection sensitivity of nanobody against aflatoxin B1 through structure-guided modification. International Journal Of Biological Macromolecules, 194, 188-197. https://doi.org/10.1016/j.ijbiomac.2021.11.182

Wang, Z., Mathias, A., Stavrou, S., & Neville, D. M. (2005). A new yeast display vector permitting free scFv amino termini can augment ligand binding affinities. Protein Engineering Design And Selection, 18(7), 337-343.