Abstract
In vivo functionalization of diatom biosilica frustules by genetic manipulation requires careful consideration of the overall structure and function of complex fusion proteins. Although we previously had transformed Thalassiosira pseudonana with constructs containing a single domain antibody (sdAb) raised against the Bacillus anthracis Sterne strain, which detected an epitope of the surface layer protein EA1 accessible in lysed spores, we initially were unsuccessful with constructs encoding a similar sdAb that detected an epitope of EA1 accessible in intact spores and vegetative cells. This discrepancy limited the usefulness of the system as an environmental biosensor for B. anthracis. We surmised that to create functional biosilica-localized biosensors with certain constructs, the biosilica targeting and protein trafficking functions of the biosilica-targeting peptide Sil3T8 had to be uncoupled. We found that retaining the ER trafficking sequence at the N-terminus and relocating the Sil3T8 targeting peptide to the C-terminus of the fusion protein resulted in successful detection of EA1 with both sdAbs. Homology modeling of antigen binding by the two sdAbs supported the hypothesis that the rescue of antigen binding in the previously dysfunctional sdAb was due to removal of steric hindrances between the antigen binding loops and the diatom biosilica for that particular sdAb.
Highlights
Diatoms are a group of unicellular microalgae, often with a highly silicified, mesoporous cell wall exhibiting nano- to meso-scale hierarchical architecture [1,2,3]
When tethering an single domain antibody (sdAb) to the diatom biosilica in this fashion, we hypothesized based on structural considerations that the antigen binding loops would be oriented hypothesized based on structural considerations that the antigen binding loops would be oriented inward and adjacent to the biosilica rather than being exposed and facing outward away from the inward and adjacent to the biosilica rather than being exposed and facing outward away from the biosilica
In order to rotate the binding loops of our sdAbs away from the biosilica surface, and thereby thereby increase antigen accessibility, the Sil3T8 tether needed to become a C-terminal fusion to the increase antigen accessibility, the Sil3T8 tether needed to become a C-terminal fusion to the sdAb
Summary
Diatoms are a group of unicellular microalgae, often with a highly silicified, mesoporous cell wall (frustule) exhibiting nano- to meso-scale hierarchical architecture [1,2,3]. VHH domain sits adjacent to the binding loops (see Figure 2 of [21], for example), we hypothesized that our existing biosilica targeting constructs with the Sil3T8 peptide fused to the N-terminus of the sdAbEA1 might produce a fusion protein whereby the antigen’s access to the sdAb binding loops was occluded. For this particular protein to be functional when tethered to diatom biosilica, fusion protein structure needed to be optimized. The biosilica functionalized with sdAbEA1 clone G10—containing fusion proteins was able to bind its target antigen EGFP-tagged EA1 protein
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call