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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.

Surface display system

In order to display the cellulosome complex on the surface of E. coli, we decided to use nanobody (Nb)-antigen (Ag) interaction between Neae-Nb3 and Ag3.

In the construction of Neae-Nb3 vector we fused ribozyme RiboJ, which is a genetic insulator that increases the protein expression of downstream sequence. The effect of RiboJ fusion was first verified using the construction of prha-riboJ-RFP plasmid (Fig.3A). Comparison of fluorescence intensity between prha-riboJ-RFP and control group absence of riboJ suggests significant increase in level of RFP expression (Fig.3C and Fig.3D).

The prha-riboJ-Neae-Nb3 plasmid were then constructed (Fig.3A) and transformed into E. coli BL21 strain for rhamnose-inducible expression. SDS-page was performed and Neae-Nb3 was presented in sediment, indicating the successful expression of Nb3 on bacterial cell surface (Fig.3B).

Figure 3: Fig.3 E. coli surface display system. (A) Construction of prha-riboJ-Neae-Nb3 and prha-riboJ-mRFP1 vectors. (B) SDS-page analysis for Neae-Nb3 expression. (C) Comparison of RFP fluorescence intensity between prha-riboJ-mRFP1 construct and prha-mRFP1 set as control. (D) Stronger positive correlation was shown between RFP fluorescence intensity and concentration of rhamnose in prha-riboJ-mRFP1 construct.

Sequence and Features

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