Optimizing the Design of Diatom Biosilica-Targeted Fusion Proteins in Biosensor Construction for Bacillus anthracis Detection
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Marine Biotechnology Group
- Western Univ. of Health Sciences, Lebanon, OR (United States). Dept. of Basic Medical Sciences
- Oregon State Univ., Corvallis, OR (United States). School of Chemical Biological and Environmental Engineering
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Marine Biotechnology Group; Oregon State Univ., Corvallis, OR (United States). School of Chemical Biological and Environmental Engineering
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.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC)
- Grant/Contract Number:
- AC05-76RL01830
- OSTI ID:
- 1628312
- Journal Information:
- Biology, Vol. 9, Issue 1; ISSN 2079-7737
- Publisher:
- MDPICopyright Statement
- Country of Publication:
- United States
- Language:
- English
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