Optimizing the Design of Diatom Biosilica-Targeted Fusion Proteins in Biosensor Construction for 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 with constructs containing a single domain antibody (sdAb) raised against the 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 .

Detection of unique Ebola virus oligonucleotides using fluorescently-labeled phosphorodiamidate morpholino oligonucleotide probe pairs.

Here we identify a low-cost diagnostic platform using fluorescently-labeled phosphorodiamidate morpholino oligonucleotide (PMO) probe pairs, which upon binding target oligonucleotides undergo fluorescence resonance energy transfer (FRET). Using a target oligonucleotide derived from the Ebola virus (EBOV), we have derivatized PMO probes with either Alexa Fluor488 (donor) or tetramethylrhodamine (acceptor). Upon EBOV target oligonulceotide binding, observed changes in FRET between PMO probe pairs permit a 25 pM lower limit of detection; there is no off-target binding within a complex mixture of nucleic acids and other biomolecules present in human saliva.

Distance-Matched Tagging Sequence Optimizes Live-Cell Protein Labeling by a Biarsenical Fluorescent Reagent AsCy3_E.

Cell permeable biarsenical fluorescent dyes built around a cyanine scaffold (AsCy3) create the ability to monitor the structural dynamics of tagged proteins in living cells. To extend the capability of this photostable and bright biarsenical probe to site-specifically label cellular proteins, we have compared the ability of AsCy3 to label two different tagging sequences (i.e.

Hydrogel Tethering Enhances Interdomain Stabilization of Single-Chain Antibodies.

Here, we identify the importance of molecular crowding agents in the functional stabilization of scFv antibodies. Antibodies were tethered through an engineered calmodulin (CaM)-binding peptide into a stimulus-responsive hydrogel composed of poly(ethylene glycol) (PEG)-functionalized CaM. Macromolecular crowding is modulated by transient heating, which decreases effective pore sizes.

Dynamic Stabilization of Expressed Proteins in Engineered Diatom Biosilica Matrices.

Self-assembly of recombinant proteins within the biosilica of living diatoms represents a means to construct functional materials in a reproducible and scalable manner that will enable applications that harness the inherent specificities of proteins to sense and respond to environmental cues. Here we describe the use of a silaffin-derived lysine-rich 39-amino-acid targeting sequence (Sil3T8) that directs a single chain fragment variable (scFv) antibody or an enhanced green fluorescent protein (EGFP) to assemble within the biosilica frustule, resulting in abundance of >200 000 proteins per frustule. Using either a fluorescent ligand bound to the scFv or the intrinsic fluorescence of EGFP, we monitored protein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability.