Optimizing the Design of Diatom Biosilica-Targeted Fusion Proteins in Biosensor Construction for <i>Bacillus anthracis</i> Detection
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 <i>Thalassiosira pseudonana</i> with constructs containing a single doma...
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| Format: | Article |
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MDPI AG
2020-01-01
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| Series: | Biology |
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| Online Access: | https://www.mdpi.com/2079-7737/9/1/14 |
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| author | Nicole R. Ford Yijia Xiong Karen A. Hecht Thomas C. Squier Gregory L. Rorrer Guritno Roesijadi |
| author_facet | Nicole R. Ford Yijia Xiong Karen A. Hecht Thomas C. Squier Gregory L. Rorrer Guritno Roesijadi |
| author_sort | Nicole R. Ford |
| collection | DOAJ |
| description | 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 <i>Thalassiosira pseudonana</i> with constructs containing a single domain antibody (sdAb) raised against the <i>Bacillus anthracis</i> 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 <i>B. anthracis</i>. We surmised that to create functional biosilica-localized biosensors with certain constructs, the biosilica targeting and protein trafficking functions of the biosilica-targeting peptide Sil3<sub>T8</sub> had to be uncoupled. We found that retaining the ER trafficking sequence at the N-terminus and relocating the Sil3<sub>T8</sub> 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. |
| first_indexed | 2024-03-12T10:58:21Z |
| format | Article |
| id | doaj.art-3d52fe080c394166ad55c2ed6d1bb130 |
| institution | Directory Open Access Journal |
| issn | 2079-7737 |
| language | English |
| last_indexed | 2024-03-12T10:58:21Z |
| publishDate | 2020-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biology |
| spelling | doaj.art-3d52fe080c394166ad55c2ed6d1bb1302023-09-02T06:06:35ZengMDPI AGBiology2079-77372020-01-01911410.3390/biology9010014biology9010014Optimizing the Design of Diatom Biosilica-Targeted Fusion Proteins in Biosensor Construction for <i>Bacillus anthracis</i> DetectionNicole R. Ford0Yijia Xiong1Karen A. Hecht2Thomas C. Squier3Gregory L. Rorrer4Guritno Roesijadi5Marine Biotechnology Group, Pacific Northwest National Laboratory, Sequim, WA 98382, USADepartment of Basic Medical Sciences, Western University of Health Sciences, Lebanon, OR 97355, USAMarine Biotechnology Group, Pacific Northwest National Laboratory, Sequim, WA 98382, USADepartment of Basic Medical Sciences, Western University of Health Sciences, Lebanon, OR 97355, USASchool of Chemical Biological and Environmental Engineering, Oregon State University, Corvallis, OR 97331, USAMarine Biotechnology Group, Pacific Northwest National Laboratory, Sequim, WA 98382, USAIn 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 <i>Thalassiosira pseudonana</i> with constructs containing a single domain antibody (sdAb) raised against the <i>Bacillus anthracis</i> 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 <i>B. anthracis</i>. We surmised that to create functional biosilica-localized biosensors with certain constructs, the biosilica targeting and protein trafficking functions of the biosilica-targeting peptide Sil3<sub>T8</sub> had to be uncoupled. We found that retaining the ER trafficking sequence at the N-terminus and relocating the Sil3<sub>T8</sub> 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.https://www.mdpi.com/2079-7737/9/1/14diatombiosilicabiosensoranthraxbiotechnologymolecular biology |
| spellingShingle | Nicole R. Ford Yijia Xiong Karen A. Hecht Thomas C. Squier Gregory L. Rorrer Guritno Roesijadi Optimizing the Design of Diatom Biosilica-Targeted Fusion Proteins in Biosensor Construction for <i>Bacillus anthracis</i> Detection Biology diatom biosilica biosensor anthrax biotechnology molecular biology |
| title | Optimizing the Design of Diatom Biosilica-Targeted Fusion Proteins in Biosensor Construction for <i>Bacillus anthracis</i> Detection |
| title_full | Optimizing the Design of Diatom Biosilica-Targeted Fusion Proteins in Biosensor Construction for <i>Bacillus anthracis</i> Detection |
| title_fullStr | Optimizing the Design of Diatom Biosilica-Targeted Fusion Proteins in Biosensor Construction for <i>Bacillus anthracis</i> Detection |
| title_full_unstemmed | Optimizing the Design of Diatom Biosilica-Targeted Fusion Proteins in Biosensor Construction for <i>Bacillus anthracis</i> Detection |
| title_short | Optimizing the Design of Diatom Biosilica-Targeted Fusion Proteins in Biosensor Construction for <i>Bacillus anthracis</i> Detection |
| title_sort | optimizing the design of diatom biosilica targeted fusion proteins in biosensor construction for i bacillus anthracis i detection |
| topic | diatom biosilica biosensor anthrax biotechnology molecular biology |
| url | https://www.mdpi.com/2079-7737/9/1/14 |
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