Creation of a Ligand-Dependent Enzyme by Fusing Circularly Permuted Antibody Variable Region Domains

Allosteric control of enzyme activity with exogenous substances has been hard to achieve, especially using antibody domains that potentially allow control by any antigens of choice. Here, in order to attain this goal, we developed a novel antibody variable region format introduced with circular permutations, called Clampbody. The two variable-region domains of the antibone Gla protein (BGP) antibody were each circularly permutated to have novel termini at the loops near their domain interface. Through their attachment to the N- and C-termini of a circularly permutated TEM-1 β-lactamase (cpBLA), we created a molecular switch that responds to the antigen peptide. The fusion protein specifically recognized the antigen, and in the presence of some detergent or denaturant, its catalytic activity was enhanced up to 4.7-fold in an antigen-dependent manner, due to increased resistance to these reagents. Hence, Clampbody will be a powerful tool for the allosteric regulation of enzyme and other protein activities and especially useful to design robust biosensors

Q‑Bodies from Recombinant Single-Chain Fv Fragment with Better Yield and Expanded Palette of Fluorophores

Fluorescence-based immunosensors serve a vital role in biotechnology and diagnostic and therapeutic applications. Our group recently developed a unique fluoroimmunosensor named Quenchbody (Q-body) that operates based on the principle of quenching and the antigen-dependent release of fluorophore, which is incorporated to a recombinant antibody fragment, either the single-chain Fv (scFv) or the Fab fragment of an antibody, using a cell-free transcription-translation system. With the objective of extending the functionality and diversity of the Q-body, here we attempted to make Q-bodies by labeling the recombinant scFv, which was prepared from E. coli using several commercially available dye-maleimides. As a result, we reproducibly obtained larger amounts of antiosteocalcin Q-bodies, with an improved yield and cost-efficiency compared with those obtained from a conventional cell-free system. The fluorescence intensity of each Q-body, including that labeled with newly tested rhodamine red, was significantly increased in the presence of an antigen with a low detection limit, although some differences in response were observed for the dye with different spacer lengths between dye and maleimide. The results indicate the Q-body’s applicability as a powerful multicolored sensor, with a potential to simultaneously monitor multiple targets in a sample

Open Flower Fluoroimmunoassay: A General Method To Make Fluorescent Protein-Based Immunosensor Probes

Fluorescence-based probes, especially those that utilize Förster resonance energy transfer (FRET) between fluorescent protein (FP) variants, are widely used to monitor various biological phenomena, most often detecting its ligand-induced conformational change through the receptor domain. While antibody provides a fertile resource of a specific receptor for various biomolecules, its potential has not been fully exploited. An exception is a pair of donor FP-fused V_H and acceptor FP-fused V_L fragments, which has been proven useful when their association increases in the presence of antigen (open sandwich fluoroimmunoassay, OS-FIA). However, probes for larger proteins such as serum albumin (SA) were difficult to produce, since the interaction between V_H and V_L of these antibodies is barely affected by the bound antigen. Here, we propose a novel strategy, called open flower fluoroimmunoassay (OF-FIA), using a probe composed of a donor-fused V_H and an acceptor-fused V_L linked by a disulfide bond between V_H and V_L (CyPet/YPet-dsFv). The probe gave high FRET efficiency due to the dimerization propensity of the FP pair, while the efficiency got lower as SA concentration increased, probably due to dimer disruption. The constructed probe could detect clinically relevant range of SA, showing its potential as a diagnostic reagent

Flashbody: A Next Generation Fluobody with Fluorescence Intensity Enhanced by Antigen Binding

Fluorescent probes are valuable tools for visualizing the spatiotemporal dynamics of molecules in living cells. Here we developed a genetically encoded antibody probe with antigen-dependent fluorescence intensity called “Flashbody”. We first created a fusion of EGFP to the single chain variable region fragment (scFv) of antibody against seven amino acids of the bone Gla protein C-terminus (BGPC7) called BGP Fluobody, which successfully showed the intracellular localization of BGPC7-tagged protein. To generate BGP Flashbody, circularly permuted GFP was inserted in between two variable region fragments, and the linkers were optimized, resulting in fluorescence intensity increase of 300% upon binding with BGPC7 in a dose-dependent manner. Live-cell imaging using BGP Flashbody showed that BGPC7 fused with cell penetrating peptide was able to enter through the plasma membrane by forming a nucleation zone, while it penetrated the nuclear membrane with different mechanism. The construction of Flashbody will be possible for a range of antibody fragments and opens up new possibilities for visualizing a myriad of molecules of interest

Apoptosis induction, caspase-8 cleavage, and the decrease in c-FLIP levels due to DX1 treatment in NB4, HP100-1 and K562 cells.

<p>(<b>A</b>) <b>Apoptotic cells.</b> HL-60, HP100-1, NB4 and K562 cells were treated with 10 µM DX1 for 9 h. The percentage of apoptotic cells was determined by morphological observation after AO/EB staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015843#s2" target="_blank">Materials and Methods</a>. (<b>B</b>) <b>The protein levels and cleavage of PARP, caspase-8 and c-FLIP.</b> NB4, HP100-1 and K562 cells were treated with or without 10 µM DX1 for 10 h. The levels of each protein were detected using specific antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015843#s2" target="_blank">Materials and Methods</a>. (<b>C</b>) <b>MG-132 enhancement of DX1-induced apoptosis.</b> Cells were treated with DMSO (control), MG-132 0.5 µM, DX1 8 µM or MG132 plus DX1 for 10 h (NB4 and HP100-1 cells) or for 36 h (K562 cells). The percentage of apoptotic cells was determined morphologically after AO/EB staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015843#s2" target="_blank">Materials and Methods</a>. * <i>P</i><0.05 compared to cells treated with DX1 alone. (<b>D</b>) <b>MG-132 enhancement of DX1-induced cleavage of PARP and caspase-8 in K562 cells.</b> K562 cells were treated with DMSO (control), MG-132 0.5 µM, DX1 8 µM or MG-132 plus DX1 for 36 h. The levels of each protein were detected using specific antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015843#s2" target="_blank">Materials and Methods</a>. (<b>E</b>) <b>The influence of c-FLIP siRNA on DX1-induced cleavage of PARP and caspase-8 in K562 cells.</b> K562 cells were incubated with control siRNA or c-FLIP siRNA for 15 h and then treated with or without 10 µM DX1 for 24 h. The levels of each protein were deteced using specific antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015843#s2" target="_blank">Materials and Methods</a>.</p

MG-132 enhanced apoptosis induction and caspase-8 cleavage due to DX1 treatment in HL-60 cells.

<p>(<b>A</b>) <b>Western blot analysis of PARP, caspase-8 and c-FLIP levels in HL-60 cells treated with MG-132.</b> HL-60 cells were treated with DMSO (control), MG-132 at the indicated concentrations for 10 h. (B) <b>Apoptotic cells determined morphologically.</b> HL-60 cells were treated with DMSO (control), MG-132 0.5 µM, DX1 8 µM or MG132 plus DX1 for 10 h. The percentage of apoptotic cells was determined by morphological observation after AO/EB staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015843#s2" target="_blank">Materials and Methods</a>. * <i>P</i><0.05 and ** <i>P</i><0.01 compared to cells treated without MG132. (<b>C</b>) <b>Western blot analysis of PARP, caspase-8, c-FLIP, DR4 and DR5 proteins.</b> HL-60 cells were treated with DMSO (control), MG-132 0.5 µM, DX1 8 µM or MG132 plus DX1 for 10 h. (<b>D</b>) <b>Time-dependent decrease of c-FLIP levels due to treatment by MG-132 plus DX1 in HL-60 cells.</b> Lane 1, control cells; Lane 2, cells treated with 0.5 µM MG-132 for 9 h; Lane 3, cells treated with 8 µM DX1 for 3 h; Lane 4, cells treated with 8 µM DX1 plus 0.5 µM MG-132 for 3 h; Lane 4, cells treated with 8 µM DX1 for 6 h; Lane 6, cells treated with 8 µM DX1 plus 0.5 µM MG-132 for 6 h; Lane 7, cells treated with 8 µM DX1 for 9 h; Lane 8, cells treated with 8 µM DX1 plus 0.5 µM MG-132 for 9 h. The levels of each protein were detected using specific antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015843#s2" target="_blank">Materials and Methods</a>.</p

Isolation of Recombinant Phage Antibodies Targeting the Hemagglutinin Cleavage Site of Highly Pathogenic Avian Influenza Virus

<div><p>Highly pathogenic avian influenza (HPAI) H5N1 viruses, which have emerged in poultry and other wildlife worldwide, contain a characteristic multi-basic cleavage site (CS) in the hemagglutinin protein (HA). Because this arginine-rich CS is unique among influenza virus subtypes, antibodies against this site have the potential to specifically diagnose pathogenic H5N1. By immunizing mice with the CS peptide and screening a phage display library, we isolated four antibody Fab fragment clones that specifically bind the antigen peptide and several HPAI H5N1 HA proteins in different clades. The soluble Fab fragments expressed in <i>Escherichia coli</i> bound the CS peptide and the H5N1 HA protein with nanomolar affinity. In an immunofluorescence assay, these Fab fragments stained cells infected with HPAI H5N1 but not those infected with a less virulent strain. Lastly, all the Fab clones could detect the CS peptide and H5N1 HA protein by open sandwich ELISA. Thus, these recombinant Fab fragments will be useful novel reagents for the rapid and specific detection of HPAI H5N1 virus.</p> </div

Purification of Fab antibodies and binding assays to rNcSAG1.

<p>SDS-polyacrylamide gel electrophoresis analysis of purified Fab antibodies (A) and the binding of A10 and H3 to rNcSAG1 proteins (B). M: Precision Plus Protein™ Dual Colors Standards. Anti-Nc ab: anti-<i>Neospora caninum</i> antibody.</p

Apoptosis induction, ROS production and the decrease in MMP by DX1 treatment in HL-60 cells.

<p>(<b>A</b>) <b>Apoptotic cell levels determined by morphological observation after AO/EB staining.</b> HL-60 cells were treated with DX1 at the indicated concentrations for 10 h or treated with DX1 at 10 µM for the indicated times. The percentage of apoptotic cells was determined morphologically using a fluorescence microscope after staining with AO and EB. The data shown are the mean plus SE of three independent experiments. (<b>B</b>) <b>Time-dependent and (C) Dose-dependent apoptosis induction due DX1 treatment determined by FACS analysis after staining with Annexin V/PI.</b> HL-60 cells were treated with DX1 at 10 µM for the indicated times (<b>B</b>) or treated with DX1 at the indicated concentrations for 10 h (<b>C</b>). The percentage of apoptotic cells was determined by FACS after staining with Annexin V. (<b>D</b>) <b>Fragmented DNA.</b> HL-60 cells were treated with DX1 at the indicated concentrations for 10 h and total DNA was isolated. The levels of fragmented DNA was determined using staining with EB after electrophoresis in agarose gel. M, DNA marker. (<b>E</b>) <b>H₂O₂ production.</b> HL-60 cells were labeled with 5 µM DCFH-DA fluorescent probe for 1 h and then treated with or without DX1 at the indicated concentrations for 8 h. Oxidized DCF levels were analyzed using FACS as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015843#s2" target="_blank">Materials and Methods</a>. Open peaks, untreated cells; shaded peaks, DX1-treated cells with the labeled concentrations. (<b>F</b>) <b>MMP.</b> HL-60 cells were treated with DX1 at the indicated concentrations for 8 h. Alterations of MMP were determined according to changes in fluorescence density upon Rhodamine 123 loading as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015843#s2" target="_blank">Materials and Methods</a>. Open peaks, untreated cells; shaded peaks, treated cells. The peak shift to left indicates a loss of MMP.</p

The structures, the antiproliferative and cytotoxic effects of β-elemene piperazine derivatives in HL-60 cells.

<p>(<b>A</b>) <b>The substitutions of β-elemene piperazine derivatives and their IG₅₀s and IC₅₀s.</b> HL-60 cells were treated with various concentrations of these compounds for 4 days. The cell number and viability were determined. The drug concentrations that inhibited half of the cell growth (IG₅₀) or that killed half of the cells (IC₅₀) were calculated. The data shown are the mean plus SE of three independent experiments. (<b>B</b>) <b>Antiproliferative effects of DX1.</b> (<b>C</b>) <b>Cytotoxicity of DX1 measured by trypan blue exclusion.</b> (<b>D</b>) <b>Cytotoxicity of DX1 measured by the MTT assay.</b> HL-60 cells were treated with the indicated concentrations of DX1 for the indicated times. Cell growth inhibition (<b>B</b>) and cytotoxicity (<b>C&D</b>) were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015843#s2" target="_blank">Materials and Methods</a>. The data shown are the mean plus SE of three independent experiments.</p