Journal: bioRxiv

Article Title: Baculovirus Surface Display of Hemagglutinin and Neuraminidase for Monoclonal Antibody Production

doi: 10.1101/2022.10.16.512416

Figure Lengend Snippet: A . Schematic representation of baculovirus expression constructs used for displaying HA7 and NA9. p-polh : polyhedrin promoter; 6S: GP64 signal peptide; 6H: 6X Histidine tag; TM: HA7 or NA9 transmembrane domain; 6C: GP64 cytoplasmic domain; C: NA9cytoplasmic domain; p-pag : pag promoter; DsRed : DsRed2 red fluorescence protein; B . Immunofluorescence staining determined the surface display of HA7 and NA9 on the Sf21 cell membranes. The cells were cultured on sterile chamber slides and infected by HA7-Bac, NA9-Bac, or mock for 3 days. Surface display of recombinant protein was detected using primary anti-His 6 monoclonal antibody followed by goat anti-mouse conjugated Alxa 488 antibody. C . Multifluorescent Western blot analysis confirmed the expression of HA7 or NA9 protein on baculovirus particles. HA7-Bac, NA9-Bac, and wild-type baculovirus (wt-Bac) were amplified and purified by sucrose gradient ultracentrifugation. Expression of His-tagged proteins (i.e., HA7 and NA9) and baculovirus GP64 protein was revealed by DyLight 800 (red fluorescence) and StarBright Blue 700 (green fluorescence), respectively (left) and quantified by fluorescence intensity (right). D . Detection of HA7 and NA9 protein on baculovirus envelopes by electron microscopy. The recombinant proteins were labeled by primary anti-His 6 monoclonal antibody and anti-mouse IgG conjugated with 6-nm gold particles. Bars: 50 nm.

Article Snippet: After removing the blocking buffer, the grids were incubated with anti-His 6 mAb (SeroTech) at a 1:100 dilution for 30 min. After two washes by PBS, 6-nm gold conjugated anti-mouse IgG (Sigma) was applied on the grids at a 1:50 dilution for 30 min. After three more washes by PBS, the grids were negatively stained by 2% phosphortungstic acid (Sigma) and examined by a transmission electron microscope (H-7500, Hitachi, Tokyo, Japan).

Techniques: Expressing, Construct, Fluorescence, Immunofluorescence, Staining, Cell Culture, Infection, Recombinant, Western Blot, Amplification, Purification, Electron Microscopy, Labeling

Journal: Cell reports

Article Title: Thiol-based functional mimicry of phosphorylation of the two-component system response regulator ArcA promotes pathogenesis in enteric pathogens

doi: 10.1016/j.celrep.2021.110147

Figure Lengend Snippet: (A) ArcA phosphorylation. V. cholerae overexpressing N-terminally His-tagged ArcA WT or ArcA D54A were grown aerobically (O 2 + ), microaerobically (O 2 − ), or microaerobically in the presence of 50 μM CHP. Whole-cell lysates were analyzed by Phos-Tag gel electrophoresis followed by anti-His6 western blotting to visualize ArcA phosphorylation. (B) ROS effects on ArcA repression of sdhC transcription. Wild type, Δ arcA , and arcA C173S mutants containing P sdhc -lacZ reporter plasmids were grown in AKI medium to mid-log phase and treated with or without 60 μM CHP for 1 h. β-Galactosidase activity was measured and normalized against viable cell numbers (SU, special units = β-gal unit/CFU × 10 6 ). The mean of nine datapoints from three independent assays is shown, and error bars represent the standard deviation; ****, p < 0.0001 (Student’s t test); ns, no significance. (C) EMSA assays. Purified ArcA wild type and ArcA-mutant derivative proteins were treated with DTT (R, reduced), carbamoyl phosphate (P, phosphorylated), and CHP (O, oxidized) before incubating with the 32 P-labeled sdhC promoter DNA. The reaction mixes were separated on a native gel and imaged on a Typhoon phosphorimager.

Article Snippet: Anti-His6 antibody , Rockland Immunochemicals , Cat# 200-301-382: RRID: AB_10703081.

Techniques: Phospho-proteomics, Nucleic Acid Electrophoresis, Western Blot, Activity Assay, Standard Deviation, Purification, Mutagenesis, Labeling

Journal: Cell reports

Article Title: Thiol-based functional mimicry of phosphorylation of the two-component system response regulator ArcA promotes pathogenesis in enteric pathogens

doi: 10.1016/j.celrep.2021.110147

Figure Lengend Snippet: (A and B) ArcA-ArcA interaction. E. coli BTH101 containing pKT25- arcA and pUC18c- arcA and derivatives were grown for 12 h at 37°C in LB aerobically or microaerobically (A) or microaerobically with or without 50 μM CHP (B). Single-step β-galactosidase activity assays were performed, and the LacZ unit was calculated by the formula described in . The mean of nine datapoints from three independent assays is shown, and error bars represent the standard deviation; ***, p < 0.001 (Student’s t test); ns, no significance. (C) V. cholerae containing overexpressed His6-tagged ArcA variants were grown under the virulence-inducing condition. When indicated, 50 μM CHP was added during ArcA induction. Cell lysates (normalized by optical density at 600 nm [OD 600 ]) with or without 100 mM BME were separated on a non-reducing 12% SDS-polyacrylamide gel followed by western blotting analysis using anti-His6 antibody; O-M, oxidized monomer; R-M, reduced monomer; O-D, oxidized dimer. (D) Full MS spectrum of the [M + 3H] 3+ ( m/z = 675.30) ions of the disulfide-linked ALLHFC 173 ENPGK and FC 233 GDLED. (E) Working model. ArcA in the reduced unphosphorylated state is in an inactive form as monomers. A microaerobic environment leads ArcA phosphorylation and thus ArcA-ArcA interaction, allowing target DNA binding. When cells are challenged by ROS, ArcA phosphorylation is abolished, but an intramolecular disulfide bond is formed, introducing a conformational change that retains ArcA-ArcA interaction and DNA binding.

Article Snippet: Anti-His6 antibody , Rockland Immunochemicals , Cat# 200-301-382: RRID: AB_10703081.

Techniques: Activity Assay, Standard Deviation, Western Blot, Phospho-proteomics, Binding Assay

Journal: Cell reports

Article Title: Thiol-based functional mimicry of phosphorylation of the two-component system response regulator ArcA promotes pathogenesis in enteric pathogens

doi: 10.1016/j.celrep.2021.110147

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-His6 antibody , Rockland Immunochemicals , Cat# 200-301-382: RRID: AB_10703081.

Techniques: Virus, Expressing, Plasmid Preparation, Recombinant, cDNA Synthesis, Control, Software, Sequencing