Difference between revisions of "Part:BBa K5466005"

Latest revision as of 00:36, 2 October 2024

AGA2P for yeast surface display, without N-terminal signal peptide

AGA2P enables yeast surface display at both the N- and C-terminus.

AGA2P is an extracellularly-secreted protein that It is part of the cell wall and is linked to anchorage subunit Aga1p via two disulfide bonds.

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.

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.

If you want to use Aga2P for yeast surface display, use the Aga2P signal peptide (BBa_K5466002).

Fuse your protein of interest between the signal peptide and this part, especially if it is a scFv or a sdAb. For sdAb and scFv display at the N-terminus, we recommend using a flexible linker 3x (GGGGS) (BBa_K5466003).

If you want to fuse the C-terminus with another protein, remove the stop codon.

Reference

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

Uchański, T., Zögg, T., Yin, J., Yuan, D., Wohlkönig, A., Fischer, B., Rosenbaum, D. M., Kobilka, B. K., Pardon, E., & Steyaert, J. (2019). An improved yeast surface display platform for the screening of nanobody immune libraries. Scientific Reports, 9(1). https://doi.org/10.1038/s41598-018-37212-3

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