recombinant human ace2 mouse fc protein  (Sino Biological)


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    Sino Biological recombinant human ace2 mouse fc protein
    Purification and characterization of S-ecto-spytag trimers from ExpiCHO cells. a. Size-exclusion chromatography (SEC) elution profile of S-ecto-spytag (S-ecto-spy) trimers. HisTrap affinity purified S-ecto-spy protein from 250 ml of transfected ExpiCHO cells was loaded on Superdex 200 prep-grade SEC column. S-trimers yield was ~50 mg per 1 L culture. b . Reducing SDS-PAGE (top) and BLUE NATIVE-PAGE (bottom) patterns of SEC-purified trimer fractions. The molecular weight standards (M) in kDa are shown on the left of the gels. IMAC (Immobilized Metal Affinity Chromatography, His) fraction is the material from affinity purification of culture supernatant on a HisTrap column, which was then loaded on the SEC column. c. ELISA analysis showing binding of purified S-trimers to human <t>ACE2</t> at various ACE2 concentrations.
    Recombinant Human Ace2 Mouse Fc Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant human ace2 mouse fc protein/product/Sino Biological
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant human ace2 mouse fc protein - by Bioz Stars, 2021-06
    86/100 stars

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    1) Product Images from "A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering"

    Article Title: A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering

    Journal: bioRxiv

    doi: 10.1101/2021.01.19.427310

    Purification and characterization of S-ecto-spytag trimers from ExpiCHO cells. a. Size-exclusion chromatography (SEC) elution profile of S-ecto-spytag (S-ecto-spy) trimers. HisTrap affinity purified S-ecto-spy protein from 250 ml of transfected ExpiCHO cells was loaded on Superdex 200 prep-grade SEC column. S-trimers yield was ~50 mg per 1 L culture. b . Reducing SDS-PAGE (top) and BLUE NATIVE-PAGE (bottom) patterns of SEC-purified trimer fractions. The molecular weight standards (M) in kDa are shown on the left of the gels. IMAC (Immobilized Metal Affinity Chromatography, His) fraction is the material from affinity purification of culture supernatant on a HisTrap column, which was then loaded on the SEC column. c. ELISA analysis showing binding of purified S-trimers to human ACE2 at various ACE2 concentrations.
    Figure Legend Snippet: Purification and characterization of S-ecto-spytag trimers from ExpiCHO cells. a. Size-exclusion chromatography (SEC) elution profile of S-ecto-spytag (S-ecto-spy) trimers. HisTrap affinity purified S-ecto-spy protein from 250 ml of transfected ExpiCHO cells was loaded on Superdex 200 prep-grade SEC column. S-trimers yield was ~50 mg per 1 L culture. b . Reducing SDS-PAGE (top) and BLUE NATIVE-PAGE (bottom) patterns of SEC-purified trimer fractions. The molecular weight standards (M) in kDa are shown on the left of the gels. IMAC (Immobilized Metal Affinity Chromatography, His) fraction is the material from affinity purification of culture supernatant on a HisTrap column, which was then loaded on the SEC column. c. ELISA analysis showing binding of purified S-trimers to human ACE2 at various ACE2 concentrations.

    Techniques Used: Purification, Size-exclusion Chromatography, Affinity Purification, Transfection, SDS Page, Blue Native PAGE, Molecular Weight, Affinity Chromatography, Enzyme-linked Immunosorbent Assay, Binding Assay

    Decoration of phage T4 nanoparticles with spike ectodomain trimers. a. Schematic showing the decoration of phage T4 nanoparticles with spike ectodomain trimers via Spytag-SpyCatcher bridges. b. In vitro assembly of S-trimers on T4-SpyCatcher phage at increasing ratios of S-trimer molecules to Soc binding sites (0:1 to 4:1). Phage and S-trimer were incubated at 4°C for 1 hr, followed by centrifugation to remove the unbound material. After two washes, the pellet was re-suspended in buffer and SDS-PAGE was performed. c. Representative cryo-EM images showing T4-SocΔ, T4-(Soc-SpyCatcher), and T4-(Soc-SpyCatcher)-S-trimers phages. The red arrowhead indicates the representative S-trimer displayed on phage. Bar = 100 nm. d. ELISA analysis of T4-S-trimer phage binding to ACE2 at various ACE2 concentrations. ****P
    Figure Legend Snippet: Decoration of phage T4 nanoparticles with spike ectodomain trimers. a. Schematic showing the decoration of phage T4 nanoparticles with spike ectodomain trimers via Spytag-SpyCatcher bridges. b. In vitro assembly of S-trimers on T4-SpyCatcher phage at increasing ratios of S-trimer molecules to Soc binding sites (0:1 to 4:1). Phage and S-trimer were incubated at 4°C for 1 hr, followed by centrifugation to remove the unbound material. After two washes, the pellet was re-suspended in buffer and SDS-PAGE was performed. c. Representative cryo-EM images showing T4-SocΔ, T4-(Soc-SpyCatcher), and T4-(Soc-SpyCatcher)-S-trimers phages. The red arrowhead indicates the representative S-trimer displayed on phage. Bar = 100 nm. d. ELISA analysis of T4-S-trimer phage binding to ACE2 at various ACE2 concentrations. ****P

    Techniques Used: In Vitro, Binding Assay, Incubation, Centrifugation, SDS Page, Enzyme-linked Immunosorbent Assay

    Construction and screening of various truncated SARS-CoV-2 RBDs. a. Structural models of recombinant WT RBD and various truncated RBDs bound to human ACE2. ACE2 is shown in green. The truncated RBD clones are shown in red and the WT RBD and deleted regions are shown in cyan. The Protein Data Bank (PDB) code for the SARS-CoV-2 RBD–ACE2 complex is 6M0J 34 . The truncated RBDs were generated using Chimera software. b. Solubility analysis of Soc-fused truncated RBDs after cloning and expression in E. coli under the control of the phage T7 promoter. After lysis of E. coli and centrifugation, the supernatant and pellet were analyzed by SDS-PAGE. The presence of Soc-truncated RBDs in the pellet and their absence in the supernatant demonstrated insolubility. The red arrowheads indicate the band positions of various Soc-truncated RBDs.
    Figure Legend Snippet: Construction and screening of various truncated SARS-CoV-2 RBDs. a. Structural models of recombinant WT RBD and various truncated RBDs bound to human ACE2. ACE2 is shown in green. The truncated RBD clones are shown in red and the WT RBD and deleted regions are shown in cyan. The Protein Data Bank (PDB) code for the SARS-CoV-2 RBD–ACE2 complex is 6M0J 34 . The truncated RBDs were generated using Chimera software. b. Solubility analysis of Soc-fused truncated RBDs after cloning and expression in E. coli under the control of the phage T7 promoter. After lysis of E. coli and centrifugation, the supernatant and pellet were analyzed by SDS-PAGE. The presence of Soc-truncated RBDs in the pellet and their absence in the supernatant demonstrated insolubility. The red arrowheads indicate the band positions of various Soc-truncated RBDs.

    Techniques Used: Recombinant, Clone Assay, Generated, Software, Solubility, Expressing, Lysis, Centrifugation, SDS Page

    Incorporation of various SARS-CoV-2 vaccine payloads into phage T4 nanoparticle. a. Schematic showing steps in T4 phage head morphogenesis. Mem, E. coli membrane; CTS, capsid targeting sequence. b and c. SDS-PAGE and Western Blot (WB) analysis of phage particles with IPII and IPIII deletions (IPIIΔIPIIIΔ) and NP encapsidation. Since NP has a very similar molecular size to T4 major capsid protein gp23*, an NP-specific antibody was used to detect NP. d. Structural model of viroporin-like tetrameric assembly of CoV-2 E protein 32 . The N-terminal seven residues and C-terminal ten residues are not shown due to the lack of a corresponding segment in the structural template used for homology modeling. Ee* indicates amino acids (aa) 8-12 and Ec* indicates aa 53-65. e. SDS-PAGE of Hoc deletion and Soc deletion phage (HocΔSocΔ). f. SDS-PAGE of recombinant phages displaying Ee-Hoc or Ec-Hoc fusion proteins. g. Schematic showing Soc-sRBD or Soc-SpyCatcher (SpyC) in vivo display on T4-SocΔ capsid. Soc-sRBD or Soc-SpyCatcher expression under the control of phage T7 promoter was induced by IPTG. Most of the expressed Soc-RBD was in the inclusion body (IB). Soluble Soc-sRBD (minor amount) or Soc-SpyC can be efficiently displayed on capsid. h. SDS-PAGE showing ~100 copies of Soc-sRBD displayed on T4 capsid. i. SDS-PAGE showing ~500 copies of Soc-SpyCatcher displayed on T4 capsid. j. Schematic diagram showing the solubilization and refolding of SUMO (small ubiquitin like modifiers)-RBD-Spytag inclusion body. Refolded SUMO-RBD-Spytag (rRBD) protein was efficiently displayed on T4-SpyCatcher phage via Spytag-SpyCatcher bridging. k. Display of rRBD on the T4-SpyCacher surface at increasing ratios of rRBD molecules to capsid Soc binding sites (0:1 to 2:1). RBD specific antibody was used to verify the displayed rRBD and rRBD-SpyCatcher-Soc complexes. T4* indicates T4-S-ecto-NP-Ec-SocΔ recombinant phage. Blue and red arrows indicate rRBD/complexes and Soc-SpyCatcher, respectively. l to o . Comparison of binding of T4-sRBD, and T4-rRBD phages to soluble human ACE2 receptor (l), monoclonal antibody (mAb) 1 (human IgG Clone #bcb03, Thermo Fisher) (m), mAb2 (rabbit IgG Clone #007, Sino Bio) (n), and polyclonal antibodies (pAb) (rabbit PAb, Sino Bio) (o) using BSA and T4 phage as controls. p. Comparison of binding of E. coli -produced rRBD to human ACE2 with the HEK293-produced RBD. **P
    Figure Legend Snippet: Incorporation of various SARS-CoV-2 vaccine payloads into phage T4 nanoparticle. a. Schematic showing steps in T4 phage head morphogenesis. Mem, E. coli membrane; CTS, capsid targeting sequence. b and c. SDS-PAGE and Western Blot (WB) analysis of phage particles with IPII and IPIII deletions (IPIIΔIPIIIΔ) and NP encapsidation. Since NP has a very similar molecular size to T4 major capsid protein gp23*, an NP-specific antibody was used to detect NP. d. Structural model of viroporin-like tetrameric assembly of CoV-2 E protein 32 . The N-terminal seven residues and C-terminal ten residues are not shown due to the lack of a corresponding segment in the structural template used for homology modeling. Ee* indicates amino acids (aa) 8-12 and Ec* indicates aa 53-65. e. SDS-PAGE of Hoc deletion and Soc deletion phage (HocΔSocΔ). f. SDS-PAGE of recombinant phages displaying Ee-Hoc or Ec-Hoc fusion proteins. g. Schematic showing Soc-sRBD or Soc-SpyCatcher (SpyC) in vivo display on T4-SocΔ capsid. Soc-sRBD or Soc-SpyCatcher expression under the control of phage T7 promoter was induced by IPTG. Most of the expressed Soc-RBD was in the inclusion body (IB). Soluble Soc-sRBD (minor amount) or Soc-SpyC can be efficiently displayed on capsid. h. SDS-PAGE showing ~100 copies of Soc-sRBD displayed on T4 capsid. i. SDS-PAGE showing ~500 copies of Soc-SpyCatcher displayed on T4 capsid. j. Schematic diagram showing the solubilization and refolding of SUMO (small ubiquitin like modifiers)-RBD-Spytag inclusion body. Refolded SUMO-RBD-Spytag (rRBD) protein was efficiently displayed on T4-SpyCatcher phage via Spytag-SpyCatcher bridging. k. Display of rRBD on the T4-SpyCacher surface at increasing ratios of rRBD molecules to capsid Soc binding sites (0:1 to 2:1). RBD specific antibody was used to verify the displayed rRBD and rRBD-SpyCatcher-Soc complexes. T4* indicates T4-S-ecto-NP-Ec-SocΔ recombinant phage. Blue and red arrows indicate rRBD/complexes and Soc-SpyCatcher, respectively. l to o . Comparison of binding of T4-sRBD, and T4-rRBD phages to soluble human ACE2 receptor (l), monoclonal antibody (mAb) 1 (human IgG Clone #bcb03, Thermo Fisher) (m), mAb2 (rabbit IgG Clone #007, Sino Bio) (n), and polyclonal antibodies (pAb) (rabbit PAb, Sino Bio) (o) using BSA and T4 phage as controls. p. Comparison of binding of E. coli -produced rRBD to human ACE2 with the HEK293-produced RBD. **P

    Techniques Used: Sequencing, SDS Page, Western Blot, Recombinant, In Vivo, Expressing, Binding Assay, Produced

    Binding of T4 phage-decorated S-trimers to ACE2-expressing HEK 293 cells. a. Co-sedimentation assay showing the capture of ACE2 by T4 phage-decorated S trimers. T4-S-trimer particles and ACE2 were incubated at equimolar ratio for 1 hr at 4°C, followed by high speed centrifugation. After two washes, the pellet was re-suspended in buffer and SDS-PAGE was performed. Presence of ACE2 in the pellet was found with these phage particles but not with the control phage lacking S-trimers. b. Immunofluorescence assay showing expression of ACE2 on 293 cells. Two days after ACE2 plasmid transfection, HEK293 cells were incubated with RBD, followed by anti-RBD antibody and Rhodamine-conjugated second antibody. c. Lack of binding of T4-GFP control phage (without S-trimers) to ACE2-293 cells. No difference in fluorescence was observed. The nuclei were stained with Hoechst. T4* indicates T4-(S-ecto)-RBD-NP-Ee-SocΔ.
    Figure Legend Snippet: Binding of T4 phage-decorated S-trimers to ACE2-expressing HEK 293 cells. a. Co-sedimentation assay showing the capture of ACE2 by T4 phage-decorated S trimers. T4-S-trimer particles and ACE2 were incubated at equimolar ratio for 1 hr at 4°C, followed by high speed centrifugation. After two washes, the pellet was re-suspended in buffer and SDS-PAGE was performed. Presence of ACE2 in the pellet was found with these phage particles but not with the control phage lacking S-trimers. b. Immunofluorescence assay showing expression of ACE2 on 293 cells. Two days after ACE2 plasmid transfection, HEK293 cells were incubated with RBD, followed by anti-RBD antibody and Rhodamine-conjugated second antibody. c. Lack of binding of T4-GFP control phage (without S-trimers) to ACE2-293 cells. No difference in fluorescence was observed. The nuclei were stained with Hoechst. T4* indicates T4-(S-ecto)-RBD-NP-Ee-SocΔ.

    Techniques Used: Binding Assay, Expressing, Sedimentation, Incubation, Centrifugation, SDS Page, Immunofluorescence, Plasmid Preparation, Transfection, Fluorescence, Staining

    Comparison of ACE2 and RBD-antibody binding of T4-sRBD, E.coli -rRBD, and HEK293-RBD. a to d . ACE2 and a panel of RBD-specific antibodies used for quantification by ELISA. e. Comparison of E. coli -produced rRBD and human HEK293-produced RBD using a panel of RBD-specific mAbs and pAbs. The HEK293-RBD showed much greater binding to mAb1 and mAb2 than the E. coli rRBD, while binding to pAbs was similar.
    Figure Legend Snippet: Comparison of ACE2 and RBD-antibody binding of T4-sRBD, E.coli -rRBD, and HEK293-RBD. a to d . ACE2 and a panel of RBD-specific antibodies used for quantification by ELISA. e. Comparison of E. coli -produced rRBD and human HEK293-produced RBD using a panel of RBD-specific mAbs and pAbs. The HEK293-RBD showed much greater binding to mAb1 and mAb2 than the E. coli rRBD, while binding to pAbs was similar.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Produced

    Related Articles

    Incubation:

    Article Title: Isolation of and Characterization of Neutralizing Antibodies to Covid-19 from a Large Human Naïve scFv Phage Display Library
    Article Snippet: FACS examination of hits binding to SpikeSpike expressing cell 1D8 was maintained in 1640 media. .. To examine if spike trimer was expressed, both FITC conjugated anti-FLAG (CST, cat#8146) and ACE2-mFc (Sinobiologics, cat#10108-H05H) and FITC-anti-mouse-IgG (Biolegend, cat#405305) were incubated with 1-2 million 1D8 cells following standard FACS protocol and analyzed by Beckman. .. To examine if the antibody hits from ELISA screening were still capable of binding to native spike trimer, the individual antibody hit at 5 μg/ml and/or ACE2-mFc was incubated with 1-2 million 1D8 spike+ cells and detected by anti-human-Fc PE (invitrogen, 12-4998-82) and FITC Goat anti-mouse-IgG (Biolegend, cat#405305) or APC Goat anti-mouse-IgG (Biolegend, cat# 405308) following standard FACS protocol and analyzed by Beckman FACS (CytoFLEX, CytExpert2.0).

    Article Title: Alveolar macrophage dysfunction and cytokine storm in the pathogenesis of two severe COVID-19 patients
    Article Snippet: 2.4.3 Antibody and S protein of SARS-CoV-2 labeling and detectionCells were firstly incubated with Fc blocker (Human BD Fc Block™, #564219, BD Biosciences). .. Then they were washed and used incubation for reagents in two different settings: purified SARS-CoV-2 (2019-nCoV) Spike Protein (#40591-V02H, Sino Biological, China), whereas the other with rabbit anti-human ACE2 antibodies (#21115-1-AP, Proteintech, China) in DMEM (Gibico) supplemented with 10% FBS (Gibico) for 30 minutes at room temperature, followed by goat anti-rabbit IgG (Alexa Fluor 546 Goat anti-Rabbit IgG (H+L) polyclonal antibody, ThermoFisher) as secondary antibodies according to manufactures’ instructions. .. Subsequently, cells treated with the above mentioned reagents in two experimental settings were respectively labeled simultaneously by antibodies against human CD3 (APC mouse anti-human CD3 monoclonal antibody, clone HIT3a, BD Bioscience), CD14 (FITC mouse anti-human CD14 monoclonal antibody, clone RMO52, Beckman Coulter), CD68 (PE/Cy7 mouse anti-human CD68 monoclonal antibody, clone Y1/82A, Biolegend) and human IgG (PE anti-human IgG Fc, clone M1310G05, Biolegend).

    Article Title: Immunogenicity of a DNA vaccine candidate for COVID-19
    Article Snippet: Plates were blocked overnight at 4 °C with blocking buffer (1× PBS, 0.05% Tween 20, 5% evaporated milk and 1% FBS). .. Plates were washed four times with washing buffer then incubated with full length (S1 + S2) spike protein containing a C-terminal His tag (Sino Biologics, cat. 40589-V08B1) at 10 µg mL−1 for 1 h at RT. .. Plates were washed and then serial dilutions of purified mouse IgG mixed with 0.1 µg mL−1 recombinant human ACE2 with a human Fc tag (ACE2-IgHu) were incubated for 1–2 h at RT.

    Article Title: Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry
    Article Snippet: .. Human ACE2 mutants bind viral spike protein. (A) A549 cells were transduced with ACE2 orthologs or human ACE2 mutants as indicated, incubated with the recombinant S1 domain of the SARS-CoV-2 or SARS-CoV spike protein C-terminally fused with Fc, and then stained with goat anti-human IgG (H + L) conjugated to Alexa Fluor 647 for flow cytometry analysis. (B) The A549 cells transduced with ACE2 orthologs and their variants as indicated, and the cells were incubated with full-length spike protein of SARS-CoV-2 (SinoBiological, 40589-V08B1) or SARS-CoV (SinoBiological, 40634-V08B) C-terminally fused with His tag, and then stained with Anti-HIS-PE (Miltenyi Biotec#130-120-787). ..

    Article Title: A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering
    Article Snippet: .. After blocking with PBS–5% BSA buffer, recombinant human ACE2-mouse Fc protein (Sino Biological) with a series of dilution was added and incubated for 1 hr at 37°C. .. Plates were then incubated with the secondary goat-anti-mouse IgG-HRP antibody and developed with TMB substrate.

    FACS:

    Article Title: Isolation of and Characterization of Neutralizing Antibodies to Covid-19 from a Large Human Naïve scFv Phage Display Library
    Article Snippet: FACS examination of hits binding to SpikeSpike expressing cell 1D8 was maintained in 1640 media. .. To examine if spike trimer was expressed, both FITC conjugated anti-FLAG (CST, cat#8146) and ACE2-mFc (Sinobiologics, cat#10108-H05H) and FITC-anti-mouse-IgG (Biolegend, cat#405305) were incubated with 1-2 million 1D8 cells following standard FACS protocol and analyzed by Beckman. .. To examine if the antibody hits from ELISA screening were still capable of binding to native spike trimer, the individual antibody hit at 5 μg/ml and/or ACE2-mFc was incubated with 1-2 million 1D8 spike+ cells and detected by anti-human-Fc PE (invitrogen, 12-4998-82) and FITC Goat anti-mouse-IgG (Biolegend, cat#405305) or APC Goat anti-mouse-IgG (Biolegend, cat# 405308) following standard FACS protocol and analyzed by Beckman FACS (CytoFLEX, CytExpert2.0).

    Purification:

    Article Title: Alveolar macrophage dysfunction and cytokine storm in the pathogenesis of two severe COVID-19 patients
    Article Snippet: 2.4.3 Antibody and S protein of SARS-CoV-2 labeling and detectionCells were firstly incubated with Fc blocker (Human BD Fc Block™, #564219, BD Biosciences). .. Then they were washed and used incubation for reagents in two different settings: purified SARS-CoV-2 (2019-nCoV) Spike Protein (#40591-V02H, Sino Biological, China), whereas the other with rabbit anti-human ACE2 antibodies (#21115-1-AP, Proteintech, China) in DMEM (Gibico) supplemented with 10% FBS (Gibico) for 30 minutes at room temperature, followed by goat anti-rabbit IgG (Alexa Fluor 546 Goat anti-Rabbit IgG (H+L) polyclonal antibody, ThermoFisher) as secondary antibodies according to manufactures’ instructions. .. Subsequently, cells treated with the above mentioned reagents in two experimental settings were respectively labeled simultaneously by antibodies against human CD3 (APC mouse anti-human CD3 monoclonal antibody, clone HIT3a, BD Bioscience), CD14 (FITC mouse anti-human CD14 monoclonal antibody, clone RMO52, Beckman Coulter), CD68 (PE/Cy7 mouse anti-human CD68 monoclonal antibody, clone Y1/82A, Biolegend) and human IgG (PE anti-human IgG Fc, clone M1310G05, Biolegend).

    other:

    Article Title: Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2
    Article Snippet: For the post-treatment condition, 25TCID50 (50% tissue culture infective dose) of SARS-CoV-2 was adsorbed for 2 h at 37°C, after washing the cells with 1xPBS, fresh culture medium (DMEM with 2%FBS) was added.

    Western Blot:

    Article Title: Inhibition of SARS-CoV-2 viral entry in vitro upon blocking N- and O-glycan elaboration
    Article Snippet: Final protein concentration was determined in ELISA format by using a calibrant Fc-protein of known concentration that was verified to be > 95% pure based on silver stain analysis. .. Identity of expressed protein and also viral Spike was determined using western blotting with anti-Fc (Jackson), anti-RBD (Sino Biologicals), anti-S2 (Sino Biologicals) and anti-ACE2 (R & D Systems) pAbs. .. Identity of expressed protein and also viral Spike was determined using western blotting with anti-Fc (Jackson), anti-RBD (Sino Biologicals), anti-S2 (Sino Biologicals) and anti-ACE2 (R & D Systems) pAbs.

    Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration
    Article Snippet: Final protein concentration was determined in ELISA format by using a calibrant Fc-protein of known concentration that was verified to be > 95% pure based on silver stain analysis. .. Identity of expressed protein and also viral Spike was determined using western blotting with anti-Fc (Jackson), anti-RBD (Sino Biologicals), anti-S2 (Sino Biologicals) and anti-ACE2 (R and D Systems) pAbs. .. Anti-FLAG mAb L5 was also used for western blotting of Spike-mutant virus as it carried a C-terminal FLAG tag.

    Transduction:

    Article Title: Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry
    Article Snippet: .. Human ACE2 mutants bind viral spike protein. (A) A549 cells were transduced with ACE2 orthologs or human ACE2 mutants as indicated, incubated with the recombinant S1 domain of the SARS-CoV-2 or SARS-CoV spike protein C-terminally fused with Fc, and then stained with goat anti-human IgG (H + L) conjugated to Alexa Fluor 647 for flow cytometry analysis. (B) The A549 cells transduced with ACE2 orthologs and their variants as indicated, and the cells were incubated with full-length spike protein of SARS-CoV-2 (SinoBiological, 40589-V08B1) or SARS-CoV (SinoBiological, 40634-V08B) C-terminally fused with His tag, and then stained with Anti-HIS-PE (Miltenyi Biotec#130-120-787). ..

    Recombinant:

    Article Title: Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry
    Article Snippet: .. Human ACE2 mutants bind viral spike protein. (A) A549 cells were transduced with ACE2 orthologs or human ACE2 mutants as indicated, incubated with the recombinant S1 domain of the SARS-CoV-2 or SARS-CoV spike protein C-terminally fused with Fc, and then stained with goat anti-human IgG (H + L) conjugated to Alexa Fluor 647 for flow cytometry analysis. (B) The A549 cells transduced with ACE2 orthologs and their variants as indicated, and the cells were incubated with full-length spike protein of SARS-CoV-2 (SinoBiological, 40589-V08B1) or SARS-CoV (SinoBiological, 40634-V08B) C-terminally fused with His tag, and then stained with Anti-HIS-PE (Miltenyi Biotec#130-120-787). ..

    Article Title: A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering
    Article Snippet: .. After blocking with PBS–5% BSA buffer, recombinant human ACE2-mouse Fc protein (Sino Biological) with a series of dilution was added and incubated for 1 hr at 37°C. .. Plates were then incubated with the secondary goat-anti-mouse IgG-HRP antibody and developed with TMB substrate.

    Staining:

    Article Title: Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry
    Article Snippet: .. Human ACE2 mutants bind viral spike protein. (A) A549 cells were transduced with ACE2 orthologs or human ACE2 mutants as indicated, incubated with the recombinant S1 domain of the SARS-CoV-2 or SARS-CoV spike protein C-terminally fused with Fc, and then stained with goat anti-human IgG (H + L) conjugated to Alexa Fluor 647 for flow cytometry analysis. (B) The A549 cells transduced with ACE2 orthologs and their variants as indicated, and the cells were incubated with full-length spike protein of SARS-CoV-2 (SinoBiological, 40589-V08B1) or SARS-CoV (SinoBiological, 40634-V08B) C-terminally fused with His tag, and then stained with Anti-HIS-PE (Miltenyi Biotec#130-120-787). ..

    Flow Cytometry:

    Article Title: Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry
    Article Snippet: .. Human ACE2 mutants bind viral spike protein. (A) A549 cells were transduced with ACE2 orthologs or human ACE2 mutants as indicated, incubated with the recombinant S1 domain of the SARS-CoV-2 or SARS-CoV spike protein C-terminally fused with Fc, and then stained with goat anti-human IgG (H + L) conjugated to Alexa Fluor 647 for flow cytometry analysis. (B) The A549 cells transduced with ACE2 orthologs and their variants as indicated, and the cells were incubated with full-length spike protein of SARS-CoV-2 (SinoBiological, 40589-V08B1) or SARS-CoV (SinoBiological, 40634-V08B) C-terminally fused with His tag, and then stained with Anti-HIS-PE (Miltenyi Biotec#130-120-787). ..

    Blocking Assay:

    Article Title: A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering
    Article Snippet: .. After blocking with PBS–5% BSA buffer, recombinant human ACE2-mouse Fc protein (Sino Biological) with a series of dilution was added and incubated for 1 hr at 37°C. .. Plates were then incubated with the secondary goat-anti-mouse IgG-HRP antibody and developed with TMB substrate.

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    Sino Biological recombinant human ace2 mouse fc protein
    Purification and characterization of S-ecto-spytag trimers from ExpiCHO cells. a. Size-exclusion chromatography (SEC) elution profile of S-ecto-spytag (S-ecto-spy) trimers. HisTrap affinity purified S-ecto-spy protein from 250 ml of transfected ExpiCHO cells was loaded on Superdex 200 prep-grade SEC column. S-trimers yield was ~50 mg per 1 L culture. b . Reducing SDS-PAGE (top) and BLUE NATIVE-PAGE (bottom) patterns of SEC-purified trimer fractions. The molecular weight standards (M) in kDa are shown on the left of the gels. IMAC (Immobilized Metal Affinity Chromatography, His) fraction is the material from affinity purification of culture supernatant on a HisTrap column, which was then loaded on the SEC column. c. ELISA analysis showing binding of purified S-trimers to human <t>ACE2</t> at various ACE2 concentrations.
    Recombinant Human Ace2 Mouse Fc Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant human ace2 mouse fc protein/product/Sino Biological
    Average 86 stars, based on 1 article reviews
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    95
    Sino Biological human recombinant ace2 fc tag
    INO-4800 immunized mouse and guinea pig sera compete with <t>ACE2</t> receptor for SARS-CoV-2 Spike protein binding. a Soluble ACE2 receptor binds to CoV-2 full-length spike with an EC 50 of 0.025 µg/ml. b Purified serum IgG from BALB/c mice ( n of 5 per group) after second immunization with INO-4800 yields significant competition against ACE2 receptor. Serum IgG samples from the animals were run in triplicate. c IgGs purified from n = 5 mice day 7 post second immunization with INO-4800 show significant competition against ACE2 receptor binding to SARS-CoV-2 S 1 + 2 protein. The soluble ACE2 concentration for the competition assay is ~0.1 µg ml −1 . Bars represent the mean and standard deviation of AUC for curves displayed in Supplementary Fig. 1 . d Hartley guinea pigs were immunized on Day 0 and 14 with 100 µg INO-4800 or pVAX-empty vector as described in the methods. Day 28 collected sera (diluted 1:20) was added SARS-CoV-2 coated wells prior to the addition of serial dilutions of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 S protein was measured. Sera collected from 5 INO-4800-treated and 3 pVAX-treated animals were used in this experiment. e Serial dilutions of guinea pig sera collected on day 21 were added to SARS-CoV-2 coated wells prior to the addition of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 S protein was measured. Sera collected from 4 INO-4800-treated and 5 pVAX-treated guinea pigs were used in this experiment.
    Human Recombinant Ace2 Fc Tag, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Sino Biological ace2 protein human recombinant
    Adjuvanted S and S1 immune sera exhibit high titers of SARS-CoV-2 pseudovirus neutralization and receptor binding inhibition activities. ( A – C ) Reduction percentage (%) in relative luminometer units (RLU) as a measure of luciferase activity for SARS-CoV-2 spike pseudotyped lentivirus infection in HEK293 cells expressing human <t>ACE2</t> receptor. Data were obtained from pooled sera ( n = 6 to 8) with triplicate wells. S-0.8 (y): S 0.8 µg boost sera of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant boost sera of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant boost sera of old aged mice, S1-4 (y): S1 4 µg boost immune sera of young adult mice, S1-4 + adj (y): S1 4 µg + adjuvant boost immune sera of young adult mice, S2-4 (y): S2 4 µg boost immune sera of young adult mice, S2-4 + adj (y): S2 4 µg + adjuvant boost immune sera of young adult mice, S-0.8 + adj (y, x3): S 0.8 µg + adjuvant 2nd boost immune sera of young adult mice, S-0.8 + adj (y, x3, 19W): S 0.8 µg + adjuvant immune sera collected at week 19 post 2nd boost of young adult mice, S-4 + adj (a, x3): S 4 µg + adjuvant 2nd boost sera of old aged mice. IV-0.8-10 + adj (y, x3): inactivated adjuvanted SARS-CoV-2 vaccination in young age mice (prime 0.8 µg of heat-inactivated and gamma-irradiated virus, 2 times boost with 10 µg inactivated adjuvanted SARS-CoV-2 of heat-inactivated and gamma-irradiated virus). Adj: adjuvants (MPL + QS-21, 1 µg + 10 µg). Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. ( D – F ) ACE2 receptor binding inhibition titers in pooled immune sera ( n = 6–8) with triplicate wells. Inhibition percentage (%) of hACE2 binding to RBD was measured after incubation with serially diluted immune sera in the plate precoated with hACE2 protein. Immune sera of groups are the same as in ( A – C ). Statistical significance was calculated using two-way ANOVA and a Bonferroni’s multiple-comparison test. Error bars indicate the mean ± SEM. **; p
    Ace2 Protein Human Recombinant, supplied by Sino Biological, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ace2 protein human recombinant - by Bioz Stars, 2021-06
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    Purification and characterization of S-ecto-spytag trimers from ExpiCHO cells. a. Size-exclusion chromatography (SEC) elution profile of S-ecto-spytag (S-ecto-spy) trimers. HisTrap affinity purified S-ecto-spy protein from 250 ml of transfected ExpiCHO cells was loaded on Superdex 200 prep-grade SEC column. S-trimers yield was ~50 mg per 1 L culture. b . Reducing SDS-PAGE (top) and BLUE NATIVE-PAGE (bottom) patterns of SEC-purified trimer fractions. The molecular weight standards (M) in kDa are shown on the left of the gels. IMAC (Immobilized Metal Affinity Chromatography, His) fraction is the material from affinity purification of culture supernatant on a HisTrap column, which was then loaded on the SEC column. c. ELISA analysis showing binding of purified S-trimers to human ACE2 at various ACE2 concentrations.

    Journal: bioRxiv

    Article Title: A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering

    doi: 10.1101/2021.01.19.427310

    Figure Lengend Snippet: Purification and characterization of S-ecto-spytag trimers from ExpiCHO cells. a. Size-exclusion chromatography (SEC) elution profile of S-ecto-spytag (S-ecto-spy) trimers. HisTrap affinity purified S-ecto-spy protein from 250 ml of transfected ExpiCHO cells was loaded on Superdex 200 prep-grade SEC column. S-trimers yield was ~50 mg per 1 L culture. b . Reducing SDS-PAGE (top) and BLUE NATIVE-PAGE (bottom) patterns of SEC-purified trimer fractions. The molecular weight standards (M) in kDa are shown on the left of the gels. IMAC (Immobilized Metal Affinity Chromatography, His) fraction is the material from affinity purification of culture supernatant on a HisTrap column, which was then loaded on the SEC column. c. ELISA analysis showing binding of purified S-trimers to human ACE2 at various ACE2 concentrations.

    Article Snippet: After blocking with PBS–5% BSA buffer, recombinant human ACE2-mouse Fc protein (Sino Biological) with a series of dilution was added and incubated for 1 hr at 37°C.

    Techniques: Purification, Size-exclusion Chromatography, Affinity Purification, Transfection, SDS Page, Blue Native PAGE, Molecular Weight, Affinity Chromatography, Enzyme-linked Immunosorbent Assay, Binding Assay

    Decoration of phage T4 nanoparticles with spike ectodomain trimers. a. Schematic showing the decoration of phage T4 nanoparticles with spike ectodomain trimers via Spytag-SpyCatcher bridges. b. In vitro assembly of S-trimers on T4-SpyCatcher phage at increasing ratios of S-trimer molecules to Soc binding sites (0:1 to 4:1). Phage and S-trimer were incubated at 4°C for 1 hr, followed by centrifugation to remove the unbound material. After two washes, the pellet was re-suspended in buffer and SDS-PAGE was performed. c. Representative cryo-EM images showing T4-SocΔ, T4-(Soc-SpyCatcher), and T4-(Soc-SpyCatcher)-S-trimers phages. The red arrowhead indicates the representative S-trimer displayed on phage. Bar = 100 nm. d. ELISA analysis of T4-S-trimer phage binding to ACE2 at various ACE2 concentrations. ****P

    Journal: bioRxiv

    Article Title: A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering

    doi: 10.1101/2021.01.19.427310

    Figure Lengend Snippet: Decoration of phage T4 nanoparticles with spike ectodomain trimers. a. Schematic showing the decoration of phage T4 nanoparticles with spike ectodomain trimers via Spytag-SpyCatcher bridges. b. In vitro assembly of S-trimers on T4-SpyCatcher phage at increasing ratios of S-trimer molecules to Soc binding sites (0:1 to 4:1). Phage and S-trimer were incubated at 4°C for 1 hr, followed by centrifugation to remove the unbound material. After two washes, the pellet was re-suspended in buffer and SDS-PAGE was performed. c. Representative cryo-EM images showing T4-SocΔ, T4-(Soc-SpyCatcher), and T4-(Soc-SpyCatcher)-S-trimers phages. The red arrowhead indicates the representative S-trimer displayed on phage. Bar = 100 nm. d. ELISA analysis of T4-S-trimer phage binding to ACE2 at various ACE2 concentrations. ****P

    Article Snippet: After blocking with PBS–5% BSA buffer, recombinant human ACE2-mouse Fc protein (Sino Biological) with a series of dilution was added and incubated for 1 hr at 37°C.

    Techniques: In Vitro, Binding Assay, Incubation, Centrifugation, SDS Page, Enzyme-linked Immunosorbent Assay

    Construction and screening of various truncated SARS-CoV-2 RBDs. a. Structural models of recombinant WT RBD and various truncated RBDs bound to human ACE2. ACE2 is shown in green. The truncated RBD clones are shown in red and the WT RBD and deleted regions are shown in cyan. The Protein Data Bank (PDB) code for the SARS-CoV-2 RBD–ACE2 complex is 6M0J 34 . The truncated RBDs were generated using Chimera software. b. Solubility analysis of Soc-fused truncated RBDs after cloning and expression in E. coli under the control of the phage T7 promoter. After lysis of E. coli and centrifugation, the supernatant and pellet were analyzed by SDS-PAGE. The presence of Soc-truncated RBDs in the pellet and their absence in the supernatant demonstrated insolubility. The red arrowheads indicate the band positions of various Soc-truncated RBDs.

    Journal: bioRxiv

    Article Title: A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering

    doi: 10.1101/2021.01.19.427310

    Figure Lengend Snippet: Construction and screening of various truncated SARS-CoV-2 RBDs. a. Structural models of recombinant WT RBD and various truncated RBDs bound to human ACE2. ACE2 is shown in green. The truncated RBD clones are shown in red and the WT RBD and deleted regions are shown in cyan. The Protein Data Bank (PDB) code for the SARS-CoV-2 RBD–ACE2 complex is 6M0J 34 . The truncated RBDs were generated using Chimera software. b. Solubility analysis of Soc-fused truncated RBDs after cloning and expression in E. coli under the control of the phage T7 promoter. After lysis of E. coli and centrifugation, the supernatant and pellet were analyzed by SDS-PAGE. The presence of Soc-truncated RBDs in the pellet and their absence in the supernatant demonstrated insolubility. The red arrowheads indicate the band positions of various Soc-truncated RBDs.

    Article Snippet: After blocking with PBS–5% BSA buffer, recombinant human ACE2-mouse Fc protein (Sino Biological) with a series of dilution was added and incubated for 1 hr at 37°C.

    Techniques: Recombinant, Clone Assay, Generated, Software, Solubility, Expressing, Lysis, Centrifugation, SDS Page

    Incorporation of various SARS-CoV-2 vaccine payloads into phage T4 nanoparticle. a. Schematic showing steps in T4 phage head morphogenesis. Mem, E. coli membrane; CTS, capsid targeting sequence. b and c. SDS-PAGE and Western Blot (WB) analysis of phage particles with IPII and IPIII deletions (IPIIΔIPIIIΔ) and NP encapsidation. Since NP has a very similar molecular size to T4 major capsid protein gp23*, an NP-specific antibody was used to detect NP. d. Structural model of viroporin-like tetrameric assembly of CoV-2 E protein 32 . The N-terminal seven residues and C-terminal ten residues are not shown due to the lack of a corresponding segment in the structural template used for homology modeling. Ee* indicates amino acids (aa) 8-12 and Ec* indicates aa 53-65. e. SDS-PAGE of Hoc deletion and Soc deletion phage (HocΔSocΔ). f. SDS-PAGE of recombinant phages displaying Ee-Hoc or Ec-Hoc fusion proteins. g. Schematic showing Soc-sRBD or Soc-SpyCatcher (SpyC) in vivo display on T4-SocΔ capsid. Soc-sRBD or Soc-SpyCatcher expression under the control of phage T7 promoter was induced by IPTG. Most of the expressed Soc-RBD was in the inclusion body (IB). Soluble Soc-sRBD (minor amount) or Soc-SpyC can be efficiently displayed on capsid. h. SDS-PAGE showing ~100 copies of Soc-sRBD displayed on T4 capsid. i. SDS-PAGE showing ~500 copies of Soc-SpyCatcher displayed on T4 capsid. j. Schematic diagram showing the solubilization and refolding of SUMO (small ubiquitin like modifiers)-RBD-Spytag inclusion body. Refolded SUMO-RBD-Spytag (rRBD) protein was efficiently displayed on T4-SpyCatcher phage via Spytag-SpyCatcher bridging. k. Display of rRBD on the T4-SpyCacher surface at increasing ratios of rRBD molecules to capsid Soc binding sites (0:1 to 2:1). RBD specific antibody was used to verify the displayed rRBD and rRBD-SpyCatcher-Soc complexes. T4* indicates T4-S-ecto-NP-Ec-SocΔ recombinant phage. Blue and red arrows indicate rRBD/complexes and Soc-SpyCatcher, respectively. l to o . Comparison of binding of T4-sRBD, and T4-rRBD phages to soluble human ACE2 receptor (l), monoclonal antibody (mAb) 1 (human IgG Clone #bcb03, Thermo Fisher) (m), mAb2 (rabbit IgG Clone #007, Sino Bio) (n), and polyclonal antibodies (pAb) (rabbit PAb, Sino Bio) (o) using BSA and T4 phage as controls. p. Comparison of binding of E. coli -produced rRBD to human ACE2 with the HEK293-produced RBD. **P

    Journal: bioRxiv

    Article Title: A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering

    doi: 10.1101/2021.01.19.427310

    Figure Lengend Snippet: Incorporation of various SARS-CoV-2 vaccine payloads into phage T4 nanoparticle. a. Schematic showing steps in T4 phage head morphogenesis. Mem, E. coli membrane; CTS, capsid targeting sequence. b and c. SDS-PAGE and Western Blot (WB) analysis of phage particles with IPII and IPIII deletions (IPIIΔIPIIIΔ) and NP encapsidation. Since NP has a very similar molecular size to T4 major capsid protein gp23*, an NP-specific antibody was used to detect NP. d. Structural model of viroporin-like tetrameric assembly of CoV-2 E protein 32 . The N-terminal seven residues and C-terminal ten residues are not shown due to the lack of a corresponding segment in the structural template used for homology modeling. Ee* indicates amino acids (aa) 8-12 and Ec* indicates aa 53-65. e. SDS-PAGE of Hoc deletion and Soc deletion phage (HocΔSocΔ). f. SDS-PAGE of recombinant phages displaying Ee-Hoc or Ec-Hoc fusion proteins. g. Schematic showing Soc-sRBD or Soc-SpyCatcher (SpyC) in vivo display on T4-SocΔ capsid. Soc-sRBD or Soc-SpyCatcher expression under the control of phage T7 promoter was induced by IPTG. Most of the expressed Soc-RBD was in the inclusion body (IB). Soluble Soc-sRBD (minor amount) or Soc-SpyC can be efficiently displayed on capsid. h. SDS-PAGE showing ~100 copies of Soc-sRBD displayed on T4 capsid. i. SDS-PAGE showing ~500 copies of Soc-SpyCatcher displayed on T4 capsid. j. Schematic diagram showing the solubilization and refolding of SUMO (small ubiquitin like modifiers)-RBD-Spytag inclusion body. Refolded SUMO-RBD-Spytag (rRBD) protein was efficiently displayed on T4-SpyCatcher phage via Spytag-SpyCatcher bridging. k. Display of rRBD on the T4-SpyCacher surface at increasing ratios of rRBD molecules to capsid Soc binding sites (0:1 to 2:1). RBD specific antibody was used to verify the displayed rRBD and rRBD-SpyCatcher-Soc complexes. T4* indicates T4-S-ecto-NP-Ec-SocΔ recombinant phage. Blue and red arrows indicate rRBD/complexes and Soc-SpyCatcher, respectively. l to o . Comparison of binding of T4-sRBD, and T4-rRBD phages to soluble human ACE2 receptor (l), monoclonal antibody (mAb) 1 (human IgG Clone #bcb03, Thermo Fisher) (m), mAb2 (rabbit IgG Clone #007, Sino Bio) (n), and polyclonal antibodies (pAb) (rabbit PAb, Sino Bio) (o) using BSA and T4 phage as controls. p. Comparison of binding of E. coli -produced rRBD to human ACE2 with the HEK293-produced RBD. **P

    Article Snippet: After blocking with PBS–5% BSA buffer, recombinant human ACE2-mouse Fc protein (Sino Biological) with a series of dilution was added and incubated for 1 hr at 37°C.

    Techniques: Sequencing, SDS Page, Western Blot, Recombinant, In Vivo, Expressing, Binding Assay, Produced

    Binding of T4 phage-decorated S-trimers to ACE2-expressing HEK 293 cells. a. Co-sedimentation assay showing the capture of ACE2 by T4 phage-decorated S trimers. T4-S-trimer particles and ACE2 were incubated at equimolar ratio for 1 hr at 4°C, followed by high speed centrifugation. After two washes, the pellet was re-suspended in buffer and SDS-PAGE was performed. Presence of ACE2 in the pellet was found with these phage particles but not with the control phage lacking S-trimers. b. Immunofluorescence assay showing expression of ACE2 on 293 cells. Two days after ACE2 plasmid transfection, HEK293 cells were incubated with RBD, followed by anti-RBD antibody and Rhodamine-conjugated second antibody. c. Lack of binding of T4-GFP control phage (without S-trimers) to ACE2-293 cells. No difference in fluorescence was observed. The nuclei were stained with Hoechst. T4* indicates T4-(S-ecto)-RBD-NP-Ee-SocΔ.

    Journal: bioRxiv

    Article Title: A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering

    doi: 10.1101/2021.01.19.427310

    Figure Lengend Snippet: Binding of T4 phage-decorated S-trimers to ACE2-expressing HEK 293 cells. a. Co-sedimentation assay showing the capture of ACE2 by T4 phage-decorated S trimers. T4-S-trimer particles and ACE2 were incubated at equimolar ratio for 1 hr at 4°C, followed by high speed centrifugation. After two washes, the pellet was re-suspended in buffer and SDS-PAGE was performed. Presence of ACE2 in the pellet was found with these phage particles but not with the control phage lacking S-trimers. b. Immunofluorescence assay showing expression of ACE2 on 293 cells. Two days after ACE2 plasmid transfection, HEK293 cells were incubated with RBD, followed by anti-RBD antibody and Rhodamine-conjugated second antibody. c. Lack of binding of T4-GFP control phage (without S-trimers) to ACE2-293 cells. No difference in fluorescence was observed. The nuclei were stained with Hoechst. T4* indicates T4-(S-ecto)-RBD-NP-Ee-SocΔ.

    Article Snippet: After blocking with PBS–5% BSA buffer, recombinant human ACE2-mouse Fc protein (Sino Biological) with a series of dilution was added and incubated for 1 hr at 37°C.

    Techniques: Binding Assay, Expressing, Sedimentation, Incubation, Centrifugation, SDS Page, Immunofluorescence, Plasmid Preparation, Transfection, Fluorescence, Staining

    Comparison of ACE2 and RBD-antibody binding of T4-sRBD, E.coli -rRBD, and HEK293-RBD. a to d . ACE2 and a panel of RBD-specific antibodies used for quantification by ELISA. e. Comparison of E. coli -produced rRBD and human HEK293-produced RBD using a panel of RBD-specific mAbs and pAbs. The HEK293-RBD showed much greater binding to mAb1 and mAb2 than the E. coli rRBD, while binding to pAbs was similar.

    Journal: bioRxiv

    Article Title: A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering

    doi: 10.1101/2021.01.19.427310

    Figure Lengend Snippet: Comparison of ACE2 and RBD-antibody binding of T4-sRBD, E.coli -rRBD, and HEK293-RBD. a to d . ACE2 and a panel of RBD-specific antibodies used for quantification by ELISA. e. Comparison of E. coli -produced rRBD and human HEK293-produced RBD using a panel of RBD-specific mAbs and pAbs. The HEK293-RBD showed much greater binding to mAb1 and mAb2 than the E. coli rRBD, while binding to pAbs was similar.

    Article Snippet: After blocking with PBS–5% BSA buffer, recombinant human ACE2-mouse Fc protein (Sino Biological) with a series of dilution was added and incubated for 1 hr at 37°C.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Produced

    INO-4800 immunized mouse and guinea pig sera compete with ACE2 receptor for SARS-CoV-2 Spike protein binding. a Soluble ACE2 receptor binds to CoV-2 full-length spike with an EC 50 of 0.025 µg/ml. b Purified serum IgG from BALB/c mice ( n of 5 per group) after second immunization with INO-4800 yields significant competition against ACE2 receptor. Serum IgG samples from the animals were run in triplicate. c IgGs purified from n = 5 mice day 7 post second immunization with INO-4800 show significant competition against ACE2 receptor binding to SARS-CoV-2 S 1 + 2 protein. The soluble ACE2 concentration for the competition assay is ~0.1 µg ml −1 . Bars represent the mean and standard deviation of AUC for curves displayed in Supplementary Fig. 1 . d Hartley guinea pigs were immunized on Day 0 and 14 with 100 µg INO-4800 or pVAX-empty vector as described in the methods. Day 28 collected sera (diluted 1:20) was added SARS-CoV-2 coated wells prior to the addition of serial dilutions of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 S protein was measured. Sera collected from 5 INO-4800-treated and 3 pVAX-treated animals were used in this experiment. e Serial dilutions of guinea pig sera collected on day 21 were added to SARS-CoV-2 coated wells prior to the addition of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 S protein was measured. Sera collected from 4 INO-4800-treated and 5 pVAX-treated guinea pigs were used in this experiment.

    Journal: Nature Communications

    Article Title: Immunogenicity of a DNA vaccine candidate for COVID-19

    doi: 10.1038/s41467-020-16505-0

    Figure Lengend Snippet: INO-4800 immunized mouse and guinea pig sera compete with ACE2 receptor for SARS-CoV-2 Spike protein binding. a Soluble ACE2 receptor binds to CoV-2 full-length spike with an EC 50 of 0.025 µg/ml. b Purified serum IgG from BALB/c mice ( n of 5 per group) after second immunization with INO-4800 yields significant competition against ACE2 receptor. Serum IgG samples from the animals were run in triplicate. c IgGs purified from n = 5 mice day 7 post second immunization with INO-4800 show significant competition against ACE2 receptor binding to SARS-CoV-2 S 1 + 2 protein. The soluble ACE2 concentration for the competition assay is ~0.1 µg ml −1 . Bars represent the mean and standard deviation of AUC for curves displayed in Supplementary Fig. 1 . d Hartley guinea pigs were immunized on Day 0 and 14 with 100 µg INO-4800 or pVAX-empty vector as described in the methods. Day 28 collected sera (diluted 1:20) was added SARS-CoV-2 coated wells prior to the addition of serial dilutions of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 S protein was measured. Sera collected from 5 INO-4800-treated and 3 pVAX-treated animals were used in this experiment. e Serial dilutions of guinea pig sera collected on day 21 were added to SARS-CoV-2 coated wells prior to the addition of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 S protein was measured. Sera collected from 4 INO-4800-treated and 5 pVAX-treated guinea pigs were used in this experiment.

    Article Snippet: Human recombinant ACE2-Fc-tag (Sinobiological) was added directly to the diluted serum, followed by 1 h of incubation at 37 °C.

    Techniques: Protein Binding, Purification, Mouse Assay, Binding Assay, Concentration Assay, Competitive Binding Assay, Standard Deviation, Plasmid Preparation

    Neutralizing antibody responses after immunization of INO-4800. BALB/c mice ( n of 5 per group) were immunized twice on days 0 and 14 with 10 µg of INO-4800. Sera was collected on day 7 post-second immunization and serial dilutions were incubated with a pseudovirus displaying the SARS-CoV-2 Spike and co-incubated with ACE2–293T cells. a Neutralization ID50 (mean ± SD) in naïve and INO-4800 immunized mice and b relative luminescence units (RLU) for sera from naive mice (green) and mice vaccinated with INO-4800 (red) as described in “Methods”.

    Journal: Nature Communications

    Article Title: Immunogenicity of a DNA vaccine candidate for COVID-19

    doi: 10.1038/s41467-020-16505-0

    Figure Lengend Snippet: Neutralizing antibody responses after immunization of INO-4800. BALB/c mice ( n of 5 per group) were immunized twice on days 0 and 14 with 10 µg of INO-4800. Sera was collected on day 7 post-second immunization and serial dilutions were incubated with a pseudovirus displaying the SARS-CoV-2 Spike and co-incubated with ACE2–293T cells. a Neutralization ID50 (mean ± SD) in naïve and INO-4800 immunized mice and b relative luminescence units (RLU) for sera from naive mice (green) and mice vaccinated with INO-4800 (red) as described in “Methods”.

    Article Snippet: Human recombinant ACE2-Fc-tag (Sinobiological) was added directly to the diluted serum, followed by 1 h of incubation at 37 °C.

    Techniques: Mouse Assay, Incubation, Neutralization

    Adjuvanted S and S1 immune sera exhibit high titers of SARS-CoV-2 pseudovirus neutralization and receptor binding inhibition activities. ( A – C ) Reduction percentage (%) in relative luminometer units (RLU) as a measure of luciferase activity for SARS-CoV-2 spike pseudotyped lentivirus infection in HEK293 cells expressing human ACE2 receptor. Data were obtained from pooled sera ( n = 6 to 8) with triplicate wells. S-0.8 (y): S 0.8 µg boost sera of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant boost sera of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant boost sera of old aged mice, S1-4 (y): S1 4 µg boost immune sera of young adult mice, S1-4 + adj (y): S1 4 µg + adjuvant boost immune sera of young adult mice, S2-4 (y): S2 4 µg boost immune sera of young adult mice, S2-4 + adj (y): S2 4 µg + adjuvant boost immune sera of young adult mice, S-0.8 + adj (y, x3): S 0.8 µg + adjuvant 2nd boost immune sera of young adult mice, S-0.8 + adj (y, x3, 19W): S 0.8 µg + adjuvant immune sera collected at week 19 post 2nd boost of young adult mice, S-4 + adj (a, x3): S 4 µg + adjuvant 2nd boost sera of old aged mice. IV-0.8-10 + adj (y, x3): inactivated adjuvanted SARS-CoV-2 vaccination in young age mice (prime 0.8 µg of heat-inactivated and gamma-irradiated virus, 2 times boost with 10 µg inactivated adjuvanted SARS-CoV-2 of heat-inactivated and gamma-irradiated virus). Adj: adjuvants (MPL + QS-21, 1 µg + 10 µg). Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. ( D – F ) ACE2 receptor binding inhibition titers in pooled immune sera ( n = 6–8) with triplicate wells. Inhibition percentage (%) of hACE2 binding to RBD was measured after incubation with serially diluted immune sera in the plate precoated with hACE2 protein. Immune sera of groups are the same as in ( A – C ). Statistical significance was calculated using two-way ANOVA and a Bonferroni’s multiple-comparison test. Error bars indicate the mean ± SEM. **; p

    Journal: Vaccines

    Article Title: Immunogenicity and Neutralizing Activity Comparison of SARS-CoV-2 Spike Full-Length and Subunit Domain Proteins in Young Adult and Old-Aged Mice

    doi: 10.3390/vaccines9040316

    Figure Lengend Snippet: Adjuvanted S and S1 immune sera exhibit high titers of SARS-CoV-2 pseudovirus neutralization and receptor binding inhibition activities. ( A – C ) Reduction percentage (%) in relative luminometer units (RLU) as a measure of luciferase activity for SARS-CoV-2 spike pseudotyped lentivirus infection in HEK293 cells expressing human ACE2 receptor. Data were obtained from pooled sera ( n = 6 to 8) with triplicate wells. S-0.8 (y): S 0.8 µg boost sera of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant boost sera of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant boost sera of old aged mice, S1-4 (y): S1 4 µg boost immune sera of young adult mice, S1-4 + adj (y): S1 4 µg + adjuvant boost immune sera of young adult mice, S2-4 (y): S2 4 µg boost immune sera of young adult mice, S2-4 + adj (y): S2 4 µg + adjuvant boost immune sera of young adult mice, S-0.8 + adj (y, x3): S 0.8 µg + adjuvant 2nd boost immune sera of young adult mice, S-0.8 + adj (y, x3, 19W): S 0.8 µg + adjuvant immune sera collected at week 19 post 2nd boost of young adult mice, S-4 + adj (a, x3): S 4 µg + adjuvant 2nd boost sera of old aged mice. IV-0.8-10 + adj (y, x3): inactivated adjuvanted SARS-CoV-2 vaccination in young age mice (prime 0.8 µg of heat-inactivated and gamma-irradiated virus, 2 times boost with 10 µg inactivated adjuvanted SARS-CoV-2 of heat-inactivated and gamma-irradiated virus). Adj: adjuvants (MPL + QS-21, 1 µg + 10 µg). Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. ( D – F ) ACE2 receptor binding inhibition titers in pooled immune sera ( n = 6–8) with triplicate wells. Inhibition percentage (%) of hACE2 binding to RBD was measured after incubation with serially diluted immune sera in the plate precoated with hACE2 protein. Immune sera of groups are the same as in ( A – C ). Statistical significance was calculated using two-way ANOVA and a Bonferroni’s multiple-comparison test. Error bars indicate the mean ± SEM. **; p

    Article Snippet: Recombinant Proteins and ReagentsSARS-CoV-2 different recombinant S and receptor proteins were obtained from Sino Biologicals (Wayne, PA, USA): Full-length S (S1–S2) ectodomain amino acid (aa) residues 16-1213 (40589-V08B1, 134.36 kDa, expressed in baculovirus-insect cells), S1 subunit (aa 16-685) with RBD domain (40591-V08H, 76.5 kDa, expressed in HEK293 cells); S2 subunit (aa 686-1213) with fusion domain (40589-V08B1, 59.36 kDa, expressed in baculovirus-insect cells); human angiotensin-converting enzyme 2 (hACE2) protein (aa 1-740) fused to Fc tag (10108-H02H, expressed in HEK293 cells).

    Techniques: Neutralization, Binding Assay, Inhibition, Luciferase, Activity Assay, Infection, Expressing, Mouse Assay, Irradiation, Incubation

    Concentration-dependent inhibition of SARS-CoV-S1S2 binding to ACE2 by representative compounds of the present study. Concentration–response curves obtained for the inhibition of the PPI between SARS-CoV-S1S2 (His-tagged, 1 μg/mL) and hACE2 (Fc-conjugated, 1 μg/mL) in cell-free ELISA-type assay by selected representative dye and nondye compounds. Data and fit as before ( Figure 3 ). Most compounds including several DRI-C compounds show similar activity against SARS-CoV (i.e., SARS-CoV-1) as against SARS-CoV-2 raising the possibility of broad-spectrum activity.

    Journal: ACS Infectious Diseases

    Article Title: Small-Molecule Inhibitors of the Coronavirus Spike: ACE2 Protein–Protein Interaction as Blockers of Viral Attachment and Entry for SARS-CoV-2

    doi: 10.1021/acsinfecdis.1c00070

    Figure Lengend Snippet: Concentration-dependent inhibition of SARS-CoV-S1S2 binding to ACE2 by representative compounds of the present study. Concentration–response curves obtained for the inhibition of the PPI between SARS-CoV-S1S2 (His-tagged, 1 μg/mL) and hACE2 (Fc-conjugated, 1 μg/mL) in cell-free ELISA-type assay by selected representative dye and nondye compounds. Data and fit as before ( Figure 3 ). Most compounds including several DRI-C compounds show similar activity against SARS-CoV (i.e., SARS-CoV-1) as against SARS-CoV-2 raising the possibility of broad-spectrum activity.

    Article Snippet: Binding Assays SARS-CoV-2 S1 and RBD (cat. no. 40591-V08H and 40592-V08H), SARS-CoV S1+S2 (cat. no. 40634-V08B), HCoV-NL63 S1 (cat. no. 40600-V08H; all with His tag), and ACE2-Fc (cat. no. 10108-H05H) used in the binding assay were obtained from SinoBiological (Wayne, PA, USA).

    Techniques: Concentration Assay, Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

    Concentration-dependent inhibition of TNF-R1–TNFα binding by compounds of the present study and corresponding selectivity plot. (A) Concentration–response curves obtained for the inhibition of this important TNF superfamily PPI in similar cell-free ELISA-type assay as used for the CoV-S–ACE2 PPIs to assess selectivity. Data and fit as before ( Figure 3 ). As the IC 50 values indicate, some of the DRI-C compounds showed more than 100-fold selectivity in inhibiting the CoV-S PPI vs the TNF PPI. (B) Selectivity plot comparing inhibitory activity (as quantified by log IC 50 ) against the TNF-R1–TNF-α interaction with that against the desired PPI target (SARS-CoV-2-S-RBD–hACE2). Active and selective compounds are clustered in the lower right corner as highlighted by the trend-indicating arrows.

    Journal: ACS Infectious Diseases

    Article Title: Small-Molecule Inhibitors of the Coronavirus Spike: ACE2 Protein–Protein Interaction as Blockers of Viral Attachment and Entry for SARS-CoV-2

    doi: 10.1021/acsinfecdis.1c00070

    Figure Lengend Snippet: Concentration-dependent inhibition of TNF-R1–TNFα binding by compounds of the present study and corresponding selectivity plot. (A) Concentration–response curves obtained for the inhibition of this important TNF superfamily PPI in similar cell-free ELISA-type assay as used for the CoV-S–ACE2 PPIs to assess selectivity. Data and fit as before ( Figure 3 ). As the IC 50 values indicate, some of the DRI-C compounds showed more than 100-fold selectivity in inhibiting the CoV-S PPI vs the TNF PPI. (B) Selectivity plot comparing inhibitory activity (as quantified by log IC 50 ) against the TNF-R1–TNF-α interaction with that against the desired PPI target (SARS-CoV-2-S-RBD–hACE2). Active and selective compounds are clustered in the lower right corner as highlighted by the trend-indicating arrows.

    Article Snippet: Binding Assays SARS-CoV-2 S1 and RBD (cat. no. 40591-V08H and 40592-V08H), SARS-CoV S1+S2 (cat. no. 40634-V08B), HCoV-NL63 S1 (cat. no. 40600-V08H; all with His tag), and ACE2-Fc (cat. no. 10108-H05H) used in the binding assay were obtained from SinoBiological (Wayne, PA, USA).

    Techniques: Concentration Assay, Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

    Concentration–response curves for binding of CoV spike protein domains to human ACE2 in cell-free ELISA-type assays. Binding curves and corresponding EC 50 ’s are shown for SARS-CoV-2 (RBD and S1), SARS-CoV (S1 S2), and HCoV-NL63 (S1). They were obtained using Fc-conjugated hACE2 coated on the plate and His-tagged S1, S1S2, or RBD added in increasing amounts as shown with the amount bound detected using an anti-His–HRP conjugate (mean ± SD for two experiments in duplicates).

    Journal: ACS Infectious Diseases

    Article Title: Small-Molecule Inhibitors of the Coronavirus Spike: ACE2 Protein–Protein Interaction as Blockers of Viral Attachment and Entry for SARS-CoV-2

    doi: 10.1021/acsinfecdis.1c00070

    Figure Lengend Snippet: Concentration–response curves for binding of CoV spike protein domains to human ACE2 in cell-free ELISA-type assays. Binding curves and corresponding EC 50 ’s are shown for SARS-CoV-2 (RBD and S1), SARS-CoV (S1 S2), and HCoV-NL63 (S1). They were obtained using Fc-conjugated hACE2 coated on the plate and His-tagged S1, S1S2, or RBD added in increasing amounts as shown with the amount bound detected using an anti-His–HRP conjugate (mean ± SD for two experiments in duplicates).

    Article Snippet: Binding Assays SARS-CoV-2 S1 and RBD (cat. no. 40591-V08H and 40592-V08H), SARS-CoV S1+S2 (cat. no. 40634-V08B), HCoV-NL63 S1 (cat. no. 40600-V08H; all with His tag), and ACE2-Fc (cat. no. 10108-H05H) used in the binding assay were obtained from SinoBiological (Wayne, PA, USA).

    Techniques: Concentration Assay, Binding Assay, Enzyme-linked Immunosorbent Assay

    Concentration-dependent inhibition of SARS-CoV-2 pseudovirus entry (BacMam) into hACE2 expressing host cells by selected compounds. Quantification of entry of pseudoviruses bearing the SARS-CoV-2 S protein (plus green fluorescent protein reporters; BacMam-based) in ACE2 (plus red fluorescence)-expressing host cells (HEK293T). Representative images (bottom row) and their quantification for pseudovirus (green) and ACE2 expression (red) using ImageJ (top row) are shown from one experiment for CgRd and DRI-C23041 in (A) and (B), respectively; average data from three experiments fitted with typical concentration–response curves are shown in (C). The amount of green present is proportional with the number of infected cells as green fluorescence is expressed only in pseudovirus infected cells, while amount of red is proportional with the number of ACE2-expressing cells. The organic dye CgRd (A), but especially DRI-C23041 (B) showed concentration-dependent inhibition with activities corresponding to low micromolar IC 50 values, whereas hydroxychloroquine (HCQ) showed no effect (C).

    Journal: ACS Infectious Diseases

    Article Title: Small-Molecule Inhibitors of the Coronavirus Spike: ACE2 Protein–Protein Interaction as Blockers of Viral Attachment and Entry for SARS-CoV-2

    doi: 10.1021/acsinfecdis.1c00070

    Figure Lengend Snippet: Concentration-dependent inhibition of SARS-CoV-2 pseudovirus entry (BacMam) into hACE2 expressing host cells by selected compounds. Quantification of entry of pseudoviruses bearing the SARS-CoV-2 S protein (plus green fluorescent protein reporters; BacMam-based) in ACE2 (plus red fluorescence)-expressing host cells (HEK293T). Representative images (bottom row) and their quantification for pseudovirus (green) and ACE2 expression (red) using ImageJ (top row) are shown from one experiment for CgRd and DRI-C23041 in (A) and (B), respectively; average data from three experiments fitted with typical concentration–response curves are shown in (C). The amount of green present is proportional with the number of infected cells as green fluorescence is expressed only in pseudovirus infected cells, while amount of red is proportional with the number of ACE2-expressing cells. The organic dye CgRd (A), but especially DRI-C23041 (B) showed concentration-dependent inhibition with activities corresponding to low micromolar IC 50 values, whereas hydroxychloroquine (HCQ) showed no effect (C).

    Article Snippet: Binding Assays SARS-CoV-2 S1 and RBD (cat. no. 40591-V08H and 40592-V08H), SARS-CoV S1+S2 (cat. no. 40634-V08B), HCoV-NL63 S1 (cat. no. 40600-V08H; all with His tag), and ACE2-Fc (cat. no. 10108-H05H) used in the binding assay were obtained from SinoBiological (Wayne, PA, USA).

    Techniques: Concentration Assay, Inhibition, Expressing, Fluorescence, Infection

    Concentration-dependent inhibition of SARS-CoV-2 pseudovirus (VSV-Δ G ) entry into hACE2/Furin expressing host cells by selected compounds. Entry of VSV-ΔG pseudoviruses bearing the SARS-CoV-2 S protein (plus GFP reporters) in ACE2/Furin overexpressing host cells (Vero-E6) was quantified via GFP fluorescence in a live imaging system (Incucyte). CgRd and DRI-C23041 showed concentration-dependent inhibition with IC 50 values consistent with the previous assay ( Figure 7 ), whereas the negative control sunset yellow (SY FD C #6) showed no significant effect.

    Journal: ACS Infectious Diseases

    Article Title: Small-Molecule Inhibitors of the Coronavirus Spike: ACE2 Protein–Protein Interaction as Blockers of Viral Attachment and Entry for SARS-CoV-2

    doi: 10.1021/acsinfecdis.1c00070

    Figure Lengend Snippet: Concentration-dependent inhibition of SARS-CoV-2 pseudovirus (VSV-Δ G ) entry into hACE2/Furin expressing host cells by selected compounds. Entry of VSV-ΔG pseudoviruses bearing the SARS-CoV-2 S protein (plus GFP reporters) in ACE2/Furin overexpressing host cells (Vero-E6) was quantified via GFP fluorescence in a live imaging system (Incucyte). CgRd and DRI-C23041 showed concentration-dependent inhibition with IC 50 values consistent with the previous assay ( Figure 7 ), whereas the negative control sunset yellow (SY FD C #6) showed no significant effect.

    Article Snippet: Binding Assays SARS-CoV-2 S1 and RBD (cat. no. 40591-V08H and 40592-V08H), SARS-CoV S1+S2 (cat. no. 40634-V08B), HCoV-NL63 S1 (cat. no. 40600-V08H; all with His tag), and ACE2-Fc (cat. no. 10108-H05H) used in the binding assay were obtained from SinoBiological (Wayne, PA, USA).

    Techniques: Concentration Assay, Inhibition, Expressing, Fluorescence, Imaging, Negative Control

    Concentration-dependent inhibition of SARS-CoV-2-S-RBD binding to ACE2 by compounds of the present study. Concentration–response curves obtained for the inhibition of the PPI between SARS-CoV-2-RBD (His-tagged, 0.5 μg/mL) and hACE2 (Fc-conjugated, 1 μg/mL) in cell-free ELISA-type assay with dye (A) and nondye (B) compounds tested. The promiscuous PPI inhibitor erythrosine B (ErB) and the food colorant FD C yellow no. 6 (sunset yellow, SY) were included as a positive and negative controls, respectively. Data are mean ± SD from two experiments in duplicates and were fitted with standard sigmoid curves for IC 50 determination. Estimated IC 50 ’s are shown in the legend indicating that while suramin and chloroquine were completely inactive (IC 50 > 500 μM), several of our in-house compounds including organic dyes (CgRd, DV1, and others) as well as proprietary DRI-C compounds (e.g., DRI-C23041, DRI-C91005) showed promising activity, some even at submicromolar levels (IC 50

    Journal: ACS Infectious Diseases

    Article Title: Small-Molecule Inhibitors of the Coronavirus Spike: ACE2 Protein–Protein Interaction as Blockers of Viral Attachment and Entry for SARS-CoV-2

    doi: 10.1021/acsinfecdis.1c00070

    Figure Lengend Snippet: Concentration-dependent inhibition of SARS-CoV-2-S-RBD binding to ACE2 by compounds of the present study. Concentration–response curves obtained for the inhibition of the PPI between SARS-CoV-2-RBD (His-tagged, 0.5 μg/mL) and hACE2 (Fc-conjugated, 1 μg/mL) in cell-free ELISA-type assay with dye (A) and nondye (B) compounds tested. The promiscuous PPI inhibitor erythrosine B (ErB) and the food colorant FD C yellow no. 6 (sunset yellow, SY) were included as a positive and negative controls, respectively. Data are mean ± SD from two experiments in duplicates and were fitted with standard sigmoid curves for IC 50 determination. Estimated IC 50 ’s are shown in the legend indicating that while suramin and chloroquine were completely inactive (IC 50 > 500 μM), several of our in-house compounds including organic dyes (CgRd, DV1, and others) as well as proprietary DRI-C compounds (e.g., DRI-C23041, DRI-C91005) showed promising activity, some even at submicromolar levels (IC 50

    Article Snippet: Binding Assays SARS-CoV-2 S1 and RBD (cat. no. 40591-V08H and 40592-V08H), SARS-CoV S1+S2 (cat. no. 40634-V08B), HCoV-NL63 S1 (cat. no. 40600-V08H; all with His tag), and ACE2-Fc (cat. no. 10108-H05H) used in the binding assay were obtained from SinoBiological (Wayne, PA, USA).

    Techniques: Concentration Assay, Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

    Identification of the binding partner by protein thermal shift. Differential scanning fluorimetry assay indicating SARS-CoV-2 RBD and not ACE2 as the binding partner of the present SMI compounds. The presence of Congo red (top) or DRI-C23041 (bottom) at 10 μM caused clear shifts in the melting temperature of the protein for RBD as indicated by the derivatives d F /d T (left; purple vs blue line), but not for hACE2 (right) (smaller insets are normalized fluorescence F data).

    Journal: ACS Infectious Diseases

    Article Title: Small-Molecule Inhibitors of the Coronavirus Spike: ACE2 Protein–Protein Interaction as Blockers of Viral Attachment and Entry for SARS-CoV-2

    doi: 10.1021/acsinfecdis.1c00070

    Figure Lengend Snippet: Identification of the binding partner by protein thermal shift. Differential scanning fluorimetry assay indicating SARS-CoV-2 RBD and not ACE2 as the binding partner of the present SMI compounds. The presence of Congo red (top) or DRI-C23041 (bottom) at 10 μM caused clear shifts in the melting temperature of the protein for RBD as indicated by the derivatives d F /d T (left; purple vs blue line), but not for hACE2 (right) (smaller insets are normalized fluorescence F data).

    Article Snippet: Binding Assays SARS-CoV-2 S1 and RBD (cat. no. 40591-V08H and 40592-V08H), SARS-CoV S1+S2 (cat. no. 40634-V08B), HCoV-NL63 S1 (cat. no. 40600-V08H; all with His tag), and ACE2-Fc (cat. no. 10108-H05H) used in the binding assay were obtained from SinoBiological (Wayne, PA, USA).

    Techniques: Binding Assay, Fluorimetry Assay, Fluorescence