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Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms Public Deposited

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https://ir.library.oregonstate.edu/concern/articles/r494vn13t

Supporting information is available online at:  http://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00191

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  • The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins comprising either enhanced green fluorescent protein (EGFP) or single chain antibodies engineered with a tetracysteine tagging sequence. Of interest were the site specific binding of (1) the fluorescent biarsenical probe AsCy3 and AsCy3e to the tetracysteine tagged fusion proteins and (2) high and low molecular mass antigens, the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT), to biosilica-immobilized single chain antibodies. Analysis of biarsenical probe binding using fluorescence and structured illumination microscopy indicated differential colocalization with EGFP in nascent and mature biosilica, supporting the use of either EGFP or bound AsCy3 and AsCy3e in studying biosilica maturation. Large increases in the lifetime of a fluorescent analogue of TNT upon binding single chain antibodies provided a robust signal capable of discriminating binding to immobilized antibodies in the transformed frustule from nonspecific binding to the biosilica matrix. In conclusion, our results demonstrate an ability to engineer diatoms to create antibody-functionalized mesoporous silica able to selectively bind chemical and biological agents for the development of sensing platforms.
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  • Ford, N. R., Hecht, K. A., Hu, D., Orr, G., Xiong, Y., Squier, T. C., ... & Roesijadi, G. (2016). Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms. ACS Synthetic Biology, 5(3), 193-199. doi:10.1021/acssynbio.5b00191
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  • 5
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  • This research was supported by the Defense Threat Reduction Agency (G.R and T.C.S.), the Office of Naval Research (G.R. and G.L.R), and the Environmental Molecular Sciences Laboratory (EMSL) (G.R. and T.C.S). Part of the research was performed using EMSL, a national scientific user facility sponsored by the DOE's OBER and located at PNNL.
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