recombinant human ace2  (Sino Biological)


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    Name:
    ACE2 Protein Human Recombinant
    Description:
    A DNA sequence encoding the extracellular domain Met 1 Ser 740 of human ACE2 precursor NP 068576 1 was expressed with the fused Fc region of human IgG1 at the C terminus
    Catalog Number:
    10108-H02H
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    ACEH Protein Human
    Host:
    HEK293 Cells
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    Structured Review

    Sino Biological recombinant human ace2
    Humoral and cellular immune responses in rhesus macaques. ( A ) The study outline showing the vaccination regimen and sample collection timepoints. ( B ) Schematic of SARS-CoV-2 spike protein. ( C ) SARS-CoV-2 S1+S2 ECD, S1, RBD and S2 protein antigen binding of IgG in serially diluted NHP sera collected. Data represents the mean endpoint titers for each individual NHP. (D E) Pseudoneutralization assay using NHP sera, showing the presence of SARS-CoV-2 specific neutralizing antibodies against the D614 ( D ) and G614 ( E ) variants of SARS-CoV-2. ( F G ) Serum collected at Week 6 from INO-4800 vaccinated NHPs inhibited <t>ACE2</t> binding to SARS-CoV-2 Spike protein. A plate-based ACE2 competition assay ( F ) and a flow-based ACE2 competition assay ( G ) showing inhibition of ACE2 binding by NHP sera. ( H ) T cell responses were measured by IFN-γ ELISpot in PBMCs stimulated for 20h with overlapping peptide pools spanning the SARS-CoV-2 Spike protein. Bars represent the mean + SD.
    A DNA sequence encoding the extracellular domain Met 1 Ser 740 of human ACE2 precursor NP 068576 1 was expressed with the fused Fc region of human IgG1 at the C terminus
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    recombinant human ace2 - by Bioz Stars, 2021-06
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    Images

    1) Product Images from "Intradermal-delivered DNA vaccine provides anamnestic protection in a rhesus macaque SARS-CoV-2 challenge model"

    Article Title: Intradermal-delivered DNA vaccine provides anamnestic protection in a rhesus macaque SARS-CoV-2 challenge model

    Journal: bioRxiv

    doi: 10.1101/2020.07.28.225649

    Humoral and cellular immune responses in rhesus macaques. ( A ) The study outline showing the vaccination regimen and sample collection timepoints. ( B ) Schematic of SARS-CoV-2 spike protein. ( C ) SARS-CoV-2 S1+S2 ECD, S1, RBD and S2 protein antigen binding of IgG in serially diluted NHP sera collected. Data represents the mean endpoint titers for each individual NHP. (D E) Pseudoneutralization assay using NHP sera, showing the presence of SARS-CoV-2 specific neutralizing antibodies against the D614 ( D ) and G614 ( E ) variants of SARS-CoV-2. ( F G ) Serum collected at Week 6 from INO-4800 vaccinated NHPs inhibited ACE2 binding to SARS-CoV-2 Spike protein. A plate-based ACE2 competition assay ( F ) and a flow-based ACE2 competition assay ( G ) showing inhibition of ACE2 binding by NHP sera. ( H ) T cell responses were measured by IFN-γ ELISpot in PBMCs stimulated for 20h with overlapping peptide pools spanning the SARS-CoV-2 Spike protein. Bars represent the mean + SD.
    Figure Legend Snippet: Humoral and cellular immune responses in rhesus macaques. ( A ) The study outline showing the vaccination regimen and sample collection timepoints. ( B ) Schematic of SARS-CoV-2 spike protein. ( C ) SARS-CoV-2 S1+S2 ECD, S1, RBD and S2 protein antigen binding of IgG in serially diluted NHP sera collected. Data represents the mean endpoint titers for each individual NHP. (D E) Pseudoneutralization assay using NHP sera, showing the presence of SARS-CoV-2 specific neutralizing antibodies against the D614 ( D ) and G614 ( E ) variants of SARS-CoV-2. ( F G ) Serum collected at Week 6 from INO-4800 vaccinated NHPs inhibited ACE2 binding to SARS-CoV-2 Spike protein. A plate-based ACE2 competition assay ( F ) and a flow-based ACE2 competition assay ( G ) showing inhibition of ACE2 binding by NHP sera. ( H ) T cell responses were measured by IFN-γ ELISpot in PBMCs stimulated for 20h with overlapping peptide pools spanning the SARS-CoV-2 Spike protein. Bars represent the mean + SD.

    Techniques Used: Binding Assay, Competitive Binding Assay, Inhibition, Enzyme-linked Immunospot

    2) Product Images from "Immunogenicity of a DNA vaccine candidate for COVID-19"

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

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16505-0

    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.
    Figure Legend 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.

    Techniques Used: 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”.
    Figure Legend 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”.

    Techniques Used: Mouse Assay, Incubation, Neutralization

    3) Product Images from "D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity"

    Article Title: D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity

    Journal: bioRxiv

    doi: 10.1101/2020.06.20.161323

    The S-G614 protein pseudotyped virus showed increased infectivity. a HEK 293T and 293T-ACE2 (human angiotensin-converting enzyme 2) cells were infected with lentiviruses pseudotyped with VSV-G and SARS-CoV-2 S protein variants. Virus titers were quantified by RT-qPCR and adjusted to 3.8 × 10 4 copies in 50 μL to normalize input virus doses. The relative luminescence units (RLU) detected 72 h post-infection (hpi). b Inhibition of pseudoviral entry by ACE2-Ig. Pseudoviruses were pre-incubated with ACE2-Ig and added to 293T-ACE2 cells, then RLU was measured at 72 hpi. c Viral entry efficiency meditated by S variants. The RLU was measured at 24-72 hpi. d-e D614G mutation facilitates elastase-2 induced pseudoviral entry. 293T-ACE2 cells were treated with elastase for 5 min and then infected with pseudotyped viruses containing the S-D614 or S-G614 mutant in the presence of various concentrations of sivelestat sodium. RLU was measured at 72 hpi. n = 3, ±SD. * P
    Figure Legend Snippet: The S-G614 protein pseudotyped virus showed increased infectivity. a HEK 293T and 293T-ACE2 (human angiotensin-converting enzyme 2) cells were infected with lentiviruses pseudotyped with VSV-G and SARS-CoV-2 S protein variants. Virus titers were quantified by RT-qPCR and adjusted to 3.8 × 10 4 copies in 50 μL to normalize input virus doses. The relative luminescence units (RLU) detected 72 h post-infection (hpi). b Inhibition of pseudoviral entry by ACE2-Ig. Pseudoviruses were pre-incubated with ACE2-Ig and added to 293T-ACE2 cells, then RLU was measured at 72 hpi. c Viral entry efficiency meditated by S variants. The RLU was measured at 24-72 hpi. d-e D614G mutation facilitates elastase-2 induced pseudoviral entry. 293T-ACE2 cells were treated with elastase for 5 min and then infected with pseudotyped viruses containing the S-D614 or S-G614 mutant in the presence of various concentrations of sivelestat sodium. RLU was measured at 72 hpi. n = 3, ±SD. * P

    Techniques Used: Infection, Quantitative RT-PCR, Inhibition, Incubation, Mutagenesis

    4) Product Images from "Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2"

    Article Title: Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2020.604663

    Analysis of purified plant-produced ACE2-Fc (lane 1) and commercial HEK293-produced ACE2-Fc (lane 2). Coomassie-stained SDS-PAGE under reducing (A) and non-reducing conditions (B) . Western blotting analysis under reducing condition with detection using a rabbit anti-ACE2 antibody (C) and an anti-human gamma-HRP conjugated antibody (D) . Western blotting analysis under non-reducing condition probed with a rabbit anti-ACE2 antibody (E) and an anti-human gamma-HRP conjugated antibody (F) .
    Figure Legend Snippet: Analysis of purified plant-produced ACE2-Fc (lane 1) and commercial HEK293-produced ACE2-Fc (lane 2). Coomassie-stained SDS-PAGE under reducing (A) and non-reducing conditions (B) . Western blotting analysis under reducing condition with detection using a rabbit anti-ACE2 antibody (C) and an anti-human gamma-HRP conjugated antibody (D) . Western blotting analysis under non-reducing condition probed with a rabbit anti-ACE2 antibody (E) and an anti-human gamma-HRP conjugated antibody (F) .

    Techniques Used: Purification, Produced, Staining, SDS Page, Western Blot

    Expression profiles of ACE2-Fc in N. benthamiana leaves on days 2, 4, 6, 8, and 10 after agroinfiltration. Leaf necrosis (A) Quantification of plant-produced ACE2-Fc (B) . The infiltrated leaves were collected from 3 individual plants in each day post infiltration. Data were analyzed by indirect ELISA assay using ACE2-specific antibody and presented as mean ± SD of triplicates.
    Figure Legend Snippet: Expression profiles of ACE2-Fc in N. benthamiana leaves on days 2, 4, 6, 8, and 10 after agroinfiltration. Leaf necrosis (A) Quantification of plant-produced ACE2-Fc (B) . The infiltrated leaves were collected from 3 individual plants in each day post infiltration. Data were analyzed by indirect ELISA assay using ACE2-specific antibody and presented as mean ± SD of triplicates.

    Techniques Used: Expressing, Produced, Indirect ELISA

    Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the pre-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.
    Figure Legend Snippet: Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the pre-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.

    Techniques Used: Produced, Inhibition, Neutralization, Infection

    Binding activity of the plant-produced ACE2-Fc with the commercial receptor binding domain of SARS-CoV-2 (SARS-CoV-2 RBD) from Sf9 cells was analyzed by ELISA. PBS buffer and S1 protein of PEDV were used as negative controls. Data are presented as mean ± SD of triplicates.
    Figure Legend Snippet: Binding activity of the plant-produced ACE2-Fc with the commercial receptor binding domain of SARS-CoV-2 (SARS-CoV-2 RBD) from Sf9 cells was analyzed by ELISA. PBS buffer and S1 protein of PEDV were used as negative controls. Data are presented as mean ± SD of triplicates.

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

    Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the post-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.
    Figure Legend Snippet: Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the post-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.

    Techniques Used: Produced, Inhibition, Neutralization, Infection

    Schematic representation of plant expression vector pBYR2e-ACE2-Fc used in the present study (A) . Diagrammatic representation showing the binding of plant-produced ACE2-Fc with SARS-CoV-2 thereby preventing the virus entry into the host cell (B) .
    Figure Legend Snippet: Schematic representation of plant expression vector pBYR2e-ACE2-Fc used in the present study (A) . Diagrammatic representation showing the binding of plant-produced ACE2-Fc with SARS-CoV-2 thereby preventing the virus entry into the host cell (B) .

    Techniques Used: Expressing, Plasmid Preparation, Binding Assay, Produced

    5) Product Images from "Epigallocatechin Gallate from Green Tea Effectively Blocks Infection of SARS-CoV-2 and New Variants by Inhibiting Spike Binding to ACE2 Receptor"

    Article Title: Epigallocatechin Gallate from Green Tea Effectively Blocks Infection of SARS-CoV-2 and New Variants by Inhibiting Spike Binding to ACE2 Receptor

    Journal: bioRxiv

    doi: 10.1101/2021.03.17.435637

    EGCG inhibits live SARS-CoV-2 and HCoV OC43 infection. ( A ) Human lung epithelial cells (Calu-3) were treated with EGCG (50,100 μM) before or after live SARS-CoV-2 (USA isolate) infection at MOI=5. Cells were washed 3 times with pre-warmed medium to remove free virus at 1 h post-infection, and then maintained in complete medium containing EGCG for 36 h. Supernatants were collected and plaque assay was carried out using Hela/ACE2-11 cells. ( B ) Cells from ( A ) were lysed and subjected to western blot to detect SARS-CoV-2 N protein. ( C ) HEK293T-hACE2 cells were pretreated with EGCG (100 μM) for 30 min prior to live SARS-CoV-2 (USA isolate) infection at MOI=0.055 or 0.275. After 1 h virus adsorption in the presence of EGCG, the cells were washed twice with pre-warmed medium, and then maintained in complete medium containing EGCG for 48 h. SARS-CoV-2 RNA copies from culture supernatant were determined at 48 h post-infection by RT-qPCR. ( D ) HCT-8 cells (susceptible cells for HCoV OC43) were pretreated with catechins at indicated dose for 30 min prior to HCoV OC43 infection at MOI=0.1. After 2 h virus adsorption, the cells were washed twice with pre-warmed medium, and then maintain in complete medium containing EGCG. ( E ) HCT-8 cells were treated with EGCG (50,100 μM) before or after live HCoV OC43 infection at MOI=0.1. After 2 h virus adsorption, the cells were washed twice with pre-warmed medium, and then maintain in complete medium containing EGCG. Intracellular HCoV OC43 RNA were determined at day 4 post-infection by qRT-PCR. Data are shown as mean ± SD, representative of two independent experiments with 3 replicates. * P
    Figure Legend Snippet: EGCG inhibits live SARS-CoV-2 and HCoV OC43 infection. ( A ) Human lung epithelial cells (Calu-3) were treated with EGCG (50,100 μM) before or after live SARS-CoV-2 (USA isolate) infection at MOI=5. Cells were washed 3 times with pre-warmed medium to remove free virus at 1 h post-infection, and then maintained in complete medium containing EGCG for 36 h. Supernatants were collected and plaque assay was carried out using Hela/ACE2-11 cells. ( B ) Cells from ( A ) were lysed and subjected to western blot to detect SARS-CoV-2 N protein. ( C ) HEK293T-hACE2 cells were pretreated with EGCG (100 μM) for 30 min prior to live SARS-CoV-2 (USA isolate) infection at MOI=0.055 or 0.275. After 1 h virus adsorption in the presence of EGCG, the cells were washed twice with pre-warmed medium, and then maintained in complete medium containing EGCG for 48 h. SARS-CoV-2 RNA copies from culture supernatant were determined at 48 h post-infection by RT-qPCR. ( D ) HCT-8 cells (susceptible cells for HCoV OC43) were pretreated with catechins at indicated dose for 30 min prior to HCoV OC43 infection at MOI=0.1. After 2 h virus adsorption, the cells were washed twice with pre-warmed medium, and then maintain in complete medium containing EGCG. ( E ) HCT-8 cells were treated with EGCG (50,100 μM) before or after live HCoV OC43 infection at MOI=0.1. After 2 h virus adsorption, the cells were washed twice with pre-warmed medium, and then maintain in complete medium containing EGCG. Intracellular HCoV OC43 RNA were determined at day 4 post-infection by qRT-PCR. Data are shown as mean ± SD, representative of two independent experiments with 3 replicates. * P

    Techniques Used: Infection, Plaque Assay, Western Blot, Adsorption, Quantitative RT-PCR

    EGCG blocks SARS-CoV-2 S binding to ACE2. ( A-D ) A549 cells were transfected with pCAG-hACE2 plasmid. Twenty-four hours later, the cells were incubated with medium containing His-tagged SARS-CoV-2 S (full-length S, S1, S2 and RBD, 1 μg/mL) with or without EGCG (50 μM) for 6 h, and subsequently incubated with goat anti-ACE2 and mouse anti-His-Tag antibodies, following stained with donkey anti-goat IgG antibody conjugated with Alexa Fluor 594 and donkey anti-mouse IgG antibody conjugated with Alexa Fluor 488. Nuclei were stained with Hoechst. All images were obtained by confocal microscopy (Nikon A1R, Nikon, Japan). The scale bar in each panel indicates 25 μm. ( E ) VeroE6 cells were incubated with medium containing His-tagged SARS-CoV-2 S (full-length S, S1, S2 and RBD, 1 μg/mL) with or without EGCG (50 μM) for 6 h, and then washed 3 times with PBS to remove unbound protein. Cell lysate was subsequently subjected to western blot analysis to detect the binding of His-tagged S to ACE2. GAPDH was set as internal control of western blot. ( F-H ) SARS-CoV-2 S (full-length S, S1 and RBD, 1 μg/mL) was premixed with EGCG at the indicated dose, and the premixture was then incubated with VeroE6 cells for 6 h, free S protein was removed with PBS triple washing. Western blot analysis was performed to assess the binding of S protein to ACE2. ( I ) Immobilized hACE2 protein (Fc tag) was precoated at 2 μg/mL at 4°C overnight, and 3-fold serially-diluted His-tagged SARS-CoV-2 full-length S (500∼0.686 ng/mL) and RBD (100∼0.137 ng/mL) were used to test the binding affinity to ACE2 in presence or absence of EGCG (50 μM) by ELISA assay. ( J ) Immobilized hACE2 protein (Fc tag) was precoated at 2 μg/mL at 4°C overnight, His-tagged full-length S (100 ng/mL) and RBD (10 ng/mL) were premixed with EGCG at indicated dose (0∼100 μM), and then used to test the binding affinity to ACE2 by ELISA assay. The data shown are representative of two independent experiments.
    Figure Legend Snippet: EGCG blocks SARS-CoV-2 S binding to ACE2. ( A-D ) A549 cells were transfected with pCAG-hACE2 plasmid. Twenty-four hours later, the cells were incubated with medium containing His-tagged SARS-CoV-2 S (full-length S, S1, S2 and RBD, 1 μg/mL) with or without EGCG (50 μM) for 6 h, and subsequently incubated with goat anti-ACE2 and mouse anti-His-Tag antibodies, following stained with donkey anti-goat IgG antibody conjugated with Alexa Fluor 594 and donkey anti-mouse IgG antibody conjugated with Alexa Fluor 488. Nuclei were stained with Hoechst. All images were obtained by confocal microscopy (Nikon A1R, Nikon, Japan). The scale bar in each panel indicates 25 μm. ( E ) VeroE6 cells were incubated with medium containing His-tagged SARS-CoV-2 S (full-length S, S1, S2 and RBD, 1 μg/mL) with or without EGCG (50 μM) for 6 h, and then washed 3 times with PBS to remove unbound protein. Cell lysate was subsequently subjected to western blot analysis to detect the binding of His-tagged S to ACE2. GAPDH was set as internal control of western blot. ( F-H ) SARS-CoV-2 S (full-length S, S1 and RBD, 1 μg/mL) was premixed with EGCG at the indicated dose, and the premixture was then incubated with VeroE6 cells for 6 h, free S protein was removed with PBS triple washing. Western blot analysis was performed to assess the binding of S protein to ACE2. ( I ) Immobilized hACE2 protein (Fc tag) was precoated at 2 μg/mL at 4°C overnight, and 3-fold serially-diluted His-tagged SARS-CoV-2 full-length S (500∼0.686 ng/mL) and RBD (100∼0.137 ng/mL) were used to test the binding affinity to ACE2 in presence or absence of EGCG (50 μM) by ELISA assay. ( J ) Immobilized hACE2 protein (Fc tag) was precoated at 2 μg/mL at 4°C overnight, His-tagged full-length S (100 ng/mL) and RBD (10 ng/mL) were premixed with EGCG at indicated dose (0∼100 μM), and then used to test the binding affinity to ACE2 by ELISA assay. The data shown are representative of two independent experiments.

    Techniques Used: Binding Assay, Transfection, Plasmid Preparation, Incubation, Staining, Confocal Microscopy, Western Blot, Enzyme-linked Immunosorbent Assay

    Related Articles

    Purification:

    Article Title: Immunogenicity of a DNA vaccine candidate for COVID-19
    Article Snippet: 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. .. Plates were again washed and then incubated with 1:10,000 dilution of HRP conjugated anti-human IgG secondary antibody (Bethyl, cat. A80-304P) and incubated for 1 h at RT.

    Recombinant:

    Article Title: Immunogenicity of a DNA vaccine candidate for COVID-19
    Article Snippet: 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. .. Plates were again washed and then incubated with 1:10,000 dilution of HRP conjugated anti-human IgG secondary antibody (Bethyl, cat. A80-304P) and incubated for 1 h at RT.

    Article Title: The utility of native MS for understanding the mechanism of action of repurposed therapeutics in COVID-19: heparin as a disruptor of the SARS-CoV-2 interaction with its host cell receptor
    Article Snippet: .. EXPERIMENTAL SECTION The recombinant forms of human ACE2 (residues 1-740) and RBD (residues 319-541) were purchased from Sino Biological (Wayne, PA). ..

    Article Title: The Utility of Native MS for Understanding the Mechanism of Action of Repurposed Therapeutics in COVID-19: Heparin as a Disruptor of the SARS-CoV-2 Interaction with Its Host Cell Receptor
    Article Snippet: .. Experimental Section The recombinant forms of human ACE2 (residues 1–740) and RBD (residues 319–541) expressed in baculovirus/insect cells systems were purchased from Sino Biological (Wayne, PA). .. RBD expressed in E. coli was purchased from RayBiotech (Peachtree Corners, GA).

    Article Title: Intradermal-delivered DNA vaccine provides anamnestic protection in a rhesus macaque SARS-CoV-2 challenge model
    Article Snippet: Following this, the cells were washed 2x with PBS followed by staining for surelight® APC conjugated anti-his antibody (Abcam, ab72579) for 30 min on ice. .. As a positive control, spike protein was pre-incubated with recombinant human ACE2 before transferring to 293T-ACE2-GFP cells. .. Data was acquired using a BD LSRII and analyzed by FlowJo (version 10).

    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: The efficacy assessment of convalescent plasma therapy for COVID-19 patients: a multi-center case series
    Article Snippet: Receptor-binding assayInhibitory effects of the CP samples on RBD-Fc binding to receptor angiotensin-converting enzyme 2 (ACE2) were tested using an ELISA-based assay. .. Recombinant soluble human ACE2 (Sino Biological) was coated at 2 µg/ml to 96-well ELISA plates (Corning Costar) in 0.1 M carbonate buffer (pH 9.6) at 4 °C overnight. ..

    Article Title: Lectin-like Intestinal Defensin Inhibits 2019-nCoV Spike binding to ACE2
    Article Snippet: The purities and molecule masses of peptides determined by reverse-phase high performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry, respectively, were shown in Table S1. .. Biolayer interferometry (BLI) The bindings of peptides and 2019-nCoV S1 (40591-V08H, Sino Biological) to recombinant human ACE2 (10108-H02H, Sino Biological, Beijing, CHN) was measured using Forte Bio’s “Octet Red 96” BLI (Pall Life Sciences, Port Washington, NYC, US). .. ACE2 (15 μg/mL) was immobilized on AR2G biosensors activated by 1-ethyl-3-[3-dimethylaminopropy] carbodiimide hydrochloride (EDC, E1769, Sigma, Shanghai, CHN) and sulfo-N-hydroxysulfosuccinimide (s-NHS, 56485, Sigma, Shanghai, CHN).

    Incubation:

    Article Title: Immunogenicity of a DNA vaccine candidate for COVID-19
    Article Snippet: 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. .. Plates were again washed and then incubated with 1:10,000 dilution of HRP conjugated anti-human IgG secondary antibody (Bethyl, cat. A80-304P) and incubated for 1 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). ..

    Positive Control:

    Article Title: Intradermal-delivered DNA vaccine provides anamnestic protection in a rhesus macaque SARS-CoV-2 challenge model
    Article Snippet: Following this, the cells were washed 2x with PBS followed by staining for surelight® APC conjugated anti-his antibody (Abcam, ab72579) for 30 min on ice. .. As a positive control, spike protein was pre-incubated with recombinant human ACE2 before transferring to 293T-ACE2-GFP cells. .. Data was acquired using a BD LSRII and analyzed by FlowJo (version 10).

    Transferring:

    Article Title: Intradermal-delivered DNA vaccine provides anamnestic protection in a rhesus macaque SARS-CoV-2 challenge model
    Article Snippet: Following this, the cells were washed 2x with PBS followed by staining for surelight® APC conjugated anti-his antibody (Abcam, ab72579) for 30 min on ice. .. As a positive control, spike protein was pre-incubated with recombinant human ACE2 before transferring to 293T-ACE2-GFP cells. .. Data was acquired using a BD LSRII and analyzed by FlowJo (version 10).

    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). ..

    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). ..

    Enzyme-linked Immunosorbent Assay:

    Article Title: The efficacy assessment of convalescent plasma therapy for COVID-19 patients: a multi-center case series
    Article Snippet: Receptor-binding assayInhibitory effects of the CP samples on RBD-Fc binding to receptor angiotensin-converting enzyme 2 (ACE2) were tested using an ELISA-based assay. .. Recombinant soluble human ACE2 (Sino Biological) was coated at 2 µg/ml to 96-well ELISA plates (Corning Costar) in 0.1 M carbonate buffer (pH 9.6) at 4 °C overnight. ..

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    Sino Biological ace2 orthologs
    Ability of <t>ACE2</t> <t>orthologs</t> and their humanized mutants to mediate entry of SARS-CoV-2 and SARS-CoV pseudoparticles. A549 cells transduced with mouse ACE2, koala ACE2 or their humanized mutants were infected with SARS-CoV-2 (A-B) or SARS-CoV (C) pseudoparticles. Two days after infection, cells were lysed and luciferase activity determined. VSV pseudoparticles were used as the control. All infections were performed in triplicate, and the data are representative of two independent experiments (mean ± standard deviation). ns, no significance; *, P
    Ace2 Orthologs, 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
    https://www.bioz.com/result/ace2 orthologs/product/Sino Biological
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ace2 orthologs - by Bioz Stars, 2021-06
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    97
    Sino Biological anti ace2 antibody rabbit polyclonal
    SARS-CoV-2 protein antigens co-express with <t>ACE2</t> in kidney tubules. a Representative expression of angiotensin-converting enzyme-II (ACE2) in kidney tissues from COVID-19 post-mortem (case #2) was detected by immunohistochemistry. Scale bars = 100 μm. b The co-expression of ACE2 and SARS-CoV-2 NP (nucleocapsid protein) or S (spike) antigens in kidney sections from COVID-19 post-mortem (case #2), hepatitis B virus-associated membranous nephropathy (HBV-MN) and trauma victims. Arrow indicates positive tubules. Scale bars = 100 μm. Data represent one of at least three technical replication each.
    Anti Ace2 Antibody Rabbit Polyclonal, 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
    https://www.bioz.com/result/anti ace2 antibody rabbit polyclonal/product/Sino Biological
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ace2 antibody rabbit polyclonal - by Bioz Stars, 2021-06
    97/100 stars
      Buy from Supplier

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    Ability of ACE2 orthologs and their humanized mutants to mediate entry of SARS-CoV-2 and SARS-CoV pseudoparticles. A549 cells transduced with mouse ACE2, koala ACE2 or their humanized mutants were infected with SARS-CoV-2 (A-B) or SARS-CoV (C) pseudoparticles. Two days after infection, cells were lysed and luciferase activity determined. VSV pseudoparticles were used as the control. All infections were performed in triplicate, and the data are representative of two independent experiments (mean ± standard deviation). ns, no significance; *, P

    Journal: PLoS Pathogens

    Article Title: Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry

    doi: 10.1371/journal.ppat.1009392

    Figure Lengend Snippet: Ability of ACE2 orthologs and their humanized mutants to mediate entry of SARS-CoV-2 and SARS-CoV pseudoparticles. A549 cells transduced with mouse ACE2, koala ACE2 or their humanized mutants were infected with SARS-CoV-2 (A-B) or SARS-CoV (C) pseudoparticles. Two days after infection, cells were lysed and luciferase activity determined. VSV pseudoparticles were used as the control. All infections were performed in triplicate, and the data are representative of two independent experiments (mean ± standard deviation). ns, no significance; *, P

    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).

    Techniques: Transduction, Infection, Luciferase, Activity Assay, Standard Deviation

    The genetic determinants of ACE2 required for SARS-CoV-2 entry. Mouse, koala, and New World monkey ACE2 cannot serve as functional receptors to support SARS-CoV-2 entry, as determined by different genetic restrictions. Position 31 in koala ACE2 is Thr whereas that in human is Lys. Substitution of Thr with Lys in koala ACE2 allowed for binding to the SARS-CoV-2 spike and viral entry. Different from koala ACE2, the genetic restriction of mouse ACE2 His353. Lys353 in human ACE2 can hydrogen bond with Gly502 of the SARS-CoV-2 spike protein, stabilizing the ACE2-spike complex. The presence of His at this position in mouse ACE2 disrupts this interaction. However, humanization of mouse ACE2 at position 353 renders the protein supportive of SARS-CoV-2 entry. The genetic determinants of New World monkey ACE2 were localized at positons 41 and 42 as we previously reported[ 20 ]. Thus, three genetic determinants of the ability of ACE2 to serve as the SARS-CoV-2 receptor were identified by comparative analysis of ACE2 orthologs, and the receptor activities of normally non-susceptible ACE2 orthologs could be rescued by genetic modification.

    Journal: PLoS Pathogens

    Article Title: Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry

    doi: 10.1371/journal.ppat.1009392

    Figure Lengend Snippet: The genetic determinants of ACE2 required for SARS-CoV-2 entry. Mouse, koala, and New World monkey ACE2 cannot serve as functional receptors to support SARS-CoV-2 entry, as determined by different genetic restrictions. Position 31 in koala ACE2 is Thr whereas that in human is Lys. Substitution of Thr with Lys in koala ACE2 allowed for binding to the SARS-CoV-2 spike and viral entry. Different from koala ACE2, the genetic restriction of mouse ACE2 His353. Lys353 in human ACE2 can hydrogen bond with Gly502 of the SARS-CoV-2 spike protein, stabilizing the ACE2-spike complex. The presence of His at this position in mouse ACE2 disrupts this interaction. However, humanization of mouse ACE2 at position 353 renders the protein supportive of SARS-CoV-2 entry. The genetic determinants of New World monkey ACE2 were localized at positons 41 and 42 as we previously reported[ 20 ]. Thus, three genetic determinants of the ability of ACE2 to serve as the SARS-CoV-2 receptor were identified by comparative analysis of ACE2 orthologs, and the receptor activities of normally non-susceptible ACE2 orthologs could be rescued by genetic modification.

    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).

    Techniques: Functional Assay, Binding Assay, Modification

    The potential residues in ACE2 that restrict SARS-CoV-2 entry. (A) A phylogenetic tree was constructed based on the protein sequences of ACE2 orthologs by using the Neighbor-joining method conducted in program MEGA7[ 40 ]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The ACE2 sequences of these species were downloaded from NCBI, and accession numbers are shown in S1 Fig . (B) Alignment of the residues of human, koala and mouse ACE2 at the interface of ACE2 with the SARS-CoV-2 spike protein (first three rows). The restrictive residues of koala or mouse ACE2 are highlighted in red. The favorable residues of human ACE2 are highlighted in green. A series of mutant ACE2 orthologs bearing restrictive or favorable residues were constructed in this study (remaining rows). (C) Cartoon of the binding interface between human ACE2 and the SARS-CoV-2 receptor-binding domain (RBD) (PDB code: 6M0J). ACE2 and the SARS-CoV-2 RBD colored in green and cyan, respectively. Key residues (K31, Y83, and K353) discussed in this study and their interacting residues are shown as ball-and-stick representations.

    Journal: PLoS Pathogens

    Article Title: Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry

    doi: 10.1371/journal.ppat.1009392

    Figure Lengend Snippet: The potential residues in ACE2 that restrict SARS-CoV-2 entry. (A) A phylogenetic tree was constructed based on the protein sequences of ACE2 orthologs by using the Neighbor-joining method conducted in program MEGA7[ 40 ]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The ACE2 sequences of these species were downloaded from NCBI, and accession numbers are shown in S1 Fig . (B) Alignment of the residues of human, koala and mouse ACE2 at the interface of ACE2 with the SARS-CoV-2 spike protein (first three rows). The restrictive residues of koala or mouse ACE2 are highlighted in red. The favorable residues of human ACE2 are highlighted in green. A series of mutant ACE2 orthologs bearing restrictive or favorable residues were constructed in this study (remaining rows). (C) Cartoon of the binding interface between human ACE2 and the SARS-CoV-2 receptor-binding domain (RBD) (PDB code: 6M0J). ACE2 and the SARS-CoV-2 RBD colored in green and cyan, respectively. Key residues (K31, Y83, and K353) discussed in this study and their interacting residues are shown as ball-and-stick representations.

    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).

    Techniques: Construct, Mutagenesis, Binding Assay

    The capability of ACE2 mutants to facilitate viral entry in vitro . (A-B) A549 cells transduced with lentiviruses expressing ACE2 orthologs or humanized mutants were infected with SARS-CoV-2 virus (MOI = 1). Expression of the viral nucleocapsid protein was visualized by immunofluorescence microscopy. Viral nucleocapsid (N) (red), and ACE2 (green) are shown. (C) The infection was quantified by a high-content imaging system. (D) The cell culture medium was collected and then virus titer was determined by focus-forming assay. The graph shows the mean and SD (mean ± standard deviation) from two independent experiments performed in triplicate. ns, no significance; *, P

    Journal: PLoS Pathogens

    Article Title: Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry

    doi: 10.1371/journal.ppat.1009392

    Figure Lengend Snippet: The capability of ACE2 mutants to facilitate viral entry in vitro . (A-B) A549 cells transduced with lentiviruses expressing ACE2 orthologs or humanized mutants were infected with SARS-CoV-2 virus (MOI = 1). Expression of the viral nucleocapsid protein was visualized by immunofluorescence microscopy. Viral nucleocapsid (N) (red), and ACE2 (green) are shown. (C) The infection was quantified by a high-content imaging system. (D) The cell culture medium was collected and then virus titer was determined by focus-forming assay. The graph shows the mean and SD (mean ± standard deviation) from two independent experiments performed in triplicate. ns, no significance; *, P

    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).

    Techniques: In Vitro, Transduction, Expressing, Infection, Immunofluorescence, Microscopy, Imaging, Cell Culture, Focus Forming Assay, Standard Deviation

    The humanized mouse ACE2 mediate viral entry in vivo. (A) Schematic representation of the experiment timeline; (B-D) The wild-type mice were transduced by recombinant adenovirus expressing ACE2 variants for 3 days, followed by SARS-CoV-2 challenge. Mice were sacrificed at day 3 post infection (n = 5 mice per group) and lung tissues were collected for H E staining (B), immunostaining (C), and viral load titration (D). The ACE2 and viral N antigen expression were detected with anti-FLAG and anti-N serum, respectively. Representative images are shown from n = 5 mice. Scale bar, 100μm (B and C). Viral load was determined by focus-forming assay. ns, no significance; *, P

    Journal: PLoS Pathogens

    Article Title: Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry

    doi: 10.1371/journal.ppat.1009392

    Figure Lengend Snippet: The humanized mouse ACE2 mediate viral entry in vivo. (A) Schematic representation of the experiment timeline; (B-D) The wild-type mice were transduced by recombinant adenovirus expressing ACE2 variants for 3 days, followed by SARS-CoV-2 challenge. Mice were sacrificed at day 3 post infection (n = 5 mice per group) and lung tissues were collected for H E staining (B), immunostaining (C), and viral load titration (D). The ACE2 and viral N antigen expression were detected with anti-FLAG and anti-N serum, respectively. Representative images are shown from n = 5 mice. Scale bar, 100μm (B and C). Viral load was determined by focus-forming assay. ns, no significance; *, P

    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).

    Techniques: In Vivo, Mouse Assay, Recombinant, Expressing, Infection, Staining, Immunostaining, Titration, Focus Forming Assay

    ACE2 mutants bind viral spike protein. (A-B) A549 cells were transduced with ACE2 orthologs or their 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. Values are expressed as the percent of cells positive for S1-Fc among the ACE2-expressing cells (zsGreen1+ cells) and shown as the means ± SD from 3 biological replicates. This experiments were independently performed three times with similar results. (C) Representative immunoblots of A549 cells transduced with lentiviruses expressing FLAG-tagged ACE2 orthologs and humanized mutants were subjected to immunoblotting. Tubulin served as the loading control. This experiments were independently performed twice with similar results. (D) The structure of SARS-CoV RBD/ACE2 complex[ 31 ] (PDB: 2AJF) and SARS-CoV-2 RBD/ACE2[ 24 ] (PDB: 6M0J) were selected for comparison. SARS-CoV RBD, SARS-CoV-2 RBD and ACE2 are colored salmon, cyan and green, respectively. K31 of ACE2 and its adjacent residues on SARS-CoV RBD or SARS-CoV2 RBD are shown as sticks.

    Journal: PLoS Pathogens

    Article Title: Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry

    doi: 10.1371/journal.ppat.1009392

    Figure Lengend Snippet: ACE2 mutants bind viral spike protein. (A-B) A549 cells were transduced with ACE2 orthologs or their 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. Values are expressed as the percent of cells positive for S1-Fc among the ACE2-expressing cells (zsGreen1+ cells) and shown as the means ± SD from 3 biological replicates. This experiments were independently performed three times with similar results. (C) Representative immunoblots of A549 cells transduced with lentiviruses expressing FLAG-tagged ACE2 orthologs and humanized mutants were subjected to immunoblotting. Tubulin served as the loading control. This experiments were independently performed twice with similar results. (D) The structure of SARS-CoV RBD/ACE2 complex[ 31 ] (PDB: 2AJF) and SARS-CoV-2 RBD/ACE2[ 24 ] (PDB: 6M0J) were selected for comparison. SARS-CoV RBD, SARS-CoV-2 RBD and ACE2 are colored salmon, cyan and green, respectively. K31 of ACE2 and its adjacent residues on SARS-CoV RBD or SARS-CoV2 RBD are shown as sticks.

    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).

    Techniques: Transduction, Incubation, Recombinant, Staining, Flow Cytometry, Expressing, Western Blot

    Mass spectra of RBD/ACE2 solutions (5 and 2.5 μM, respectively) in 150 mM NH 4 CH 3 CO 2 acquired in the absence of heparinoids (A) and in the presence of fondaparinux (B) and dp20 (C). The reference mass spectrum of ACE2 is shown in panel A (red); the blue, pink, and purple reference spectra in panels A, B, and C, respectively, represent RBD, RBD/fondaparinux, and RBD/dp20. The well-defined charge ladders in each panel show the results of the limited charge reduction measurements that were used to assign the poorly defined ion peaks in the original mass spectra.

    Journal: Analytical Chemistry

    Article Title: The Utility of Native MS for Understanding the Mechanism of Action of Repurposed Therapeutics in COVID-19: Heparin as a Disruptor of the SARS-CoV-2 Interaction with Its Host Cell Receptor

    doi: 10.1021/acs.analchem.0c02449

    Figure Lengend Snippet: Mass spectra of RBD/ACE2 solutions (5 and 2.5 μM, respectively) in 150 mM NH 4 CH 3 CO 2 acquired in the absence of heparinoids (A) and in the presence of fondaparinux (B) and dp20 (C). The reference mass spectrum of ACE2 is shown in panel A (red); the blue, pink, and purple reference spectra in panels A, B, and C, respectively, represent RBD, RBD/fondaparinux, and RBD/dp20. The well-defined charge ladders in each panel show the results of the limited charge reduction measurements that were used to assign the poorly defined ion peaks in the original mass spectra.

    Article Snippet: Experimental Section The recombinant forms of human ACE2 (residues 1–740) and RBD (residues 319–541) expressed in baculovirus/insect cells systems were purchased from Sino Biological (Wayne, PA).

    Techniques:

    3 kT / e electrostatic potential (ESP) surfaces calculated for RBD associated with ACE2 (left), fondaparinux (middle), and dp20 (right). The ACE2/RBD structure (part of PDB 6M17( 28 )) shows ACE2 in a ribbon format with an ESP-mapped molecular surface. Both RBD/heparinoid complexes are representative structures from MD simulations; the heparinoids are shown in a ball-and-stick format with ESP-mapped molecular surfaces.

    Journal: Analytical Chemistry

    Article Title: The Utility of Native MS for Understanding the Mechanism of Action of Repurposed Therapeutics in COVID-19: Heparin as a Disruptor of the SARS-CoV-2 Interaction with Its Host Cell Receptor

    doi: 10.1021/acs.analchem.0c02449

    Figure Lengend Snippet: 3 kT / e electrostatic potential (ESP) surfaces calculated for RBD associated with ACE2 (left), fondaparinux (middle), and dp20 (right). The ACE2/RBD structure (part of PDB 6M17( 28 )) shows ACE2 in a ribbon format with an ESP-mapped molecular surface. Both RBD/heparinoid complexes are representative structures from MD simulations; the heparinoids are shown in a ball-and-stick format with ESP-mapped molecular surfaces.

    Article Snippet: Experimental Section The recombinant forms of human ACE2 (residues 1–740) and RBD (residues 319–541) expressed in baculovirus/insect cells systems were purchased from Sino Biological (Wayne, PA).

    Techniques:

    Native MS of RBD/ACE2 solutions (5 and 2.5 μM, respectively, in 150 mM NH 4 CH 3 CO 2 ) acquired in the absence of heparinoids (bottom), and in the presence of fondaparinux (middle) and dp20 (top). The faded color-filled curves show reference mass spectra of ACE2 (red), RBD (blue), RBD/fondaparinux (pink) and RBD/dp20 (purple). The well-defined charge ladders show the results if limited charge reduction measurements that were used to assign charges to poorly-defined ion peaks in the native MS.

    Journal: bioRxiv

    Article Title: The utility of native MS for understanding the mechanism of action of repurposed therapeutics in COVID-19: heparin as a disruptor of the SARS-CoV-2 interaction with its host cell receptor

    doi: 10.1101/2020.06.09.142794

    Figure Lengend Snippet: Native MS of RBD/ACE2 solutions (5 and 2.5 μM, respectively, in 150 mM NH 4 CH 3 CO 2 ) acquired in the absence of heparinoids (bottom), and in the presence of fondaparinux (middle) and dp20 (top). The faded color-filled curves show reference mass spectra of ACE2 (red), RBD (blue), RBD/fondaparinux (pink) and RBD/dp20 (purple). The well-defined charge ladders show the results if limited charge reduction measurements that were used to assign charges to poorly-defined ion peaks in the native MS.

    Article Snippet: EXPERIMENTAL SECTION The recombinant forms of human ACE2 (residues 1-740) and RBD (residues 319-541) were purchased from Sino Biological (Wayne, PA).

    Techniques:

    The 3 kT / e isopotential surfaces calculated for RBD associated with ACE2 (left), fondaparinux (middle) and dp20 (right). The RBD/ACE2 structure (part of pbd 6M17 31 ) shows ACE2 in a ribbon format (red). Both RBD/heparinoid complexes show representative structures obtained from MD simulations; both heparinoids are shown in a ball-and-stick format (oxygen and sulfur atoms are colored in red and orange, respectively).

    Journal: bioRxiv

    Article Title: The utility of native MS for understanding the mechanism of action of repurposed therapeutics in COVID-19: heparin as a disruptor of the SARS-CoV-2 interaction with its host cell receptor

    doi: 10.1101/2020.06.09.142794

    Figure Lengend Snippet: The 3 kT / e isopotential surfaces calculated for RBD associated with ACE2 (left), fondaparinux (middle) and dp20 (right). The RBD/ACE2 structure (part of pbd 6M17 31 ) shows ACE2 in a ribbon format (red). Both RBD/heparinoid complexes show representative structures obtained from MD simulations; both heparinoids are shown in a ball-and-stick format (oxygen and sulfur atoms are colored in red and orange, respectively).

    Article Snippet: EXPERIMENTAL SECTION The recombinant forms of human ACE2 (residues 1-740) and RBD (residues 319-541) were purchased from Sino Biological (Wayne, PA).

    Techniques:

    SARS-CoV-2 protein antigens co-express with ACE2 in kidney tubules. a Representative expression of angiotensin-converting enzyme-II (ACE2) in kidney tissues from COVID-19 post-mortem (case #2) was detected by immunohistochemistry. Scale bars = 100 μm. b The co-expression of ACE2 and SARS-CoV-2 NP (nucleocapsid protein) or S (spike) antigens in kidney sections from COVID-19 post-mortem (case #2), hepatitis B virus-associated membranous nephropathy (HBV-MN) and trauma victims. Arrow indicates positive tubules. Scale bars = 100 μm. Data represent one of at least three technical replication each.

    Journal: Nature Communications

    Article Title: Human kidney is a target for novel severe acute respiratory syndrome coronavirus 2 infection

    doi: 10.1038/s41467-021-22781-1

    Figure Lengend Snippet: SARS-CoV-2 protein antigens co-express with ACE2 in kidney tubules. a Representative expression of angiotensin-converting enzyme-II (ACE2) in kidney tissues from COVID-19 post-mortem (case #2) was detected by immunohistochemistry. Scale bars = 100 μm. b The co-expression of ACE2 and SARS-CoV-2 NP (nucleocapsid protein) or S (spike) antigens in kidney sections from COVID-19 post-mortem (case #2), hepatitis B virus-associated membranous nephropathy (HBV-MN) and trauma victims. Arrow indicates positive tubules. Scale bars = 100 μm. Data represent one of at least three technical replication each.

    Article Snippet: Immunofluorescence double-staining For immunofluorescence double-staining, the sections were incubated with primary mouse originated antibodies including anti-SARS NP antibodies (ab273434, 1:500, mouse IgG), anti-SARS S antibodies (ab273433, 1:500, mouse IgG), or anti-DP2 (sc-271898, 1:200, mouse monoclonal C-5; Santa Cruz Biotechnology) and rabbit originated antibodies including anti-SARS NP antibodies (clone ID: 019, 1:200, rabbit IgG; Sino Biological, Beijing), anti-ACE2 (clone ID: 10108-RP01, 1:100, rabbit IgG; Sino Biological) or anti-PDS (ab182141, 1:100, rabbit IgG1; Abcam) at 4 °C overnight, respectively.

    Techniques: Expressing, Immunohistochemistry