title: Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms.
abstract: 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.
authors.affiliation: Marine Biotechnology, Pacific Northwest National Laboratory , Sequim, Washington 98382, United States.
authors.forename: Nicole R
authors.initials: NR
authors.lastname: Ford
authors.affiliation: Marine Biotechnology, Pacific Northwest National Laboratory , Sequim, Washington 98382, United States.
authors.forename: Karen A
authors.initials: KA
authors.lastname: Hecht
authors.affiliation: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory , Richland, Washington 99352, United States.
authors.forename: DeHong
authors.initials: D
authors.lastname: Hu
authors.affiliation: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory , Richland, Washington 99352, United States.
authors.forename: Galya
authors.initials: G
authors.lastname: Orr
authors.affiliation: Department of Basic Medical Sciences, Western University of Health Sciences , Lebanon, Oregon 97355, United States.
authors.forename: Yijia
authors.initials: Y
authors.lastname: Xiong
authors.affiliation: Department of Basic Medical Sciences, Western University of Health Sciences , Lebanon, Oregon 97355, United States.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Squier
authors.forename: Gregory L
authors.initials: GL
authors.lastname: Rorrer
authors.affiliation: Marine Biotechnology, Pacific Northwest National Laboratory , Sequim, Washington 98382, United States.
authors.forename: Guritno
authors.initials: G
authors.lastname: Roesijadi
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D000941:Antigens / Q000737:chemistry / Q000378:metabolism*; D001408:Bacillus anthracis / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D017377:Diatoms / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055767:Immobilized Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012822:Silicon Dioxide / Q000737:chemistry*; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D014303:Trinitrotoluene / Q000276:immunology
chemical_list: D055910:Antibodies, Immobilized; D000941:Antigens; D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D055767:Immobilized Proteins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C485184:enhanced green fluorescent protein; D014303:Trinitrotoluene; D049452:Green Fluorescent Proteins; D012822:Silicon Dioxide
keywords: TNT; anthrax; biarsenical probe; biosensor; diatom; silica