Part:BBa_K4275026

Neae-Nb3

Neae-Nb is a type of nanobody, which contains variable domains of camelid heavy-chain antibodies, that can be expressed on bacterial surfaces due to their small size (~125 amino acids) and stability under a variety of conditions[1]. The combination between the single-domain structure and the intimin autotransporter allows the entirety of a highly specific, cell surface-bound adhesin to be encoded as a single fusion protein. Neae-Nb will specifically adhere to a corresponding antigen via the Nb-Ag interaction, which can form the adhesins and control morphology and patterning of multicellular assemblies.

Figure 1 The 3D structure of the protein predicted by Alphafold2.

Usage and Biology

Nanobodies are the recombinant variable domains of heavy-chain-only antibodies, with many unique properties such as small size, excellent solubility and superior stability. This nanobody domain interacts with specific antigen through antigen nanobody interactions and can be used for cell-cell adhesion and bacterial surface display with a high adhesion stability.The CDR(Complementarity-determining region) domain of the nanobodies determine their antigen-recognition specificity.In our experiment, Nb3[1] displayed on the outer membrane specifically interacts with Ag3 on the OlpB-Ag3 fusion construct (BBa_K4275013), promoting the immobilization of cellulosome complexes onto the surface of the bacteria.

Characterization

Nanobody-antigen interaction

An E. coli expression vector of surface display system Neae-Nb was constructed and transformed into E. coli host cells. The export tag fused with the coding sequence directs the nanobody domain to be exported and presented on the extracellular surface of the bacteria. Interaction between the exposed nanobody domain and the antigen3 domain fused with an eforRed reporter protein was shown by the red fluorescent characteristics in the pellet formed after centrifugation, which is absent in the control group only with Neae-Nb. This highly efficient cell surface adhesion approach was proven useful in a variety of fields and aid our needs for constructing complex nanomachines on the surface of E. coli.

File:Nb-Ag-eforRED.png

Figure 2: Fig.2 The surface display system characterized by nanobody-antigen interaction between Neae-Nb3 and Ag3 fused with reporter protein eforRED.

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]

References

1. Glass, David S, and Ingmar H Riedel-Kruse. “A Synthetic Bacterial Cell-Cell Adhesion Toolbox for Programming Multicellular Morphologies and Patterns.” Cell vol. 174,3 (2018): 649-658.e16. doi:10.1016/j.cell.2018.06.041