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Nb28-S102D Aga2P

Yeast surface display of Nb28-S102D with Aga2P. The localization of the nanobody on the cell wall allows the capture of AFB1 from the environment.

Sequence and Features

Usage and Biology

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 and fragments such as Fab and scFv (~15 kDa) and typically consist of only three complementarity-determining regions (CDRs). Despite this, they maintain a broad reactivity with diverse epitopes, aided by the extended flexible CDR3 loop that allows recognition of hidden or cryptic epitopes. Recent studies indicate that Nbs can form concave-shaped binding sites for small molecules, similar to conventional antibodies. Given these advantageous properties, Nbs are emerging as promising alternatives to traditional antibodies.

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

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. https://doi.org/10.1093/protein/gzi036

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