Journal: Frontiers in Microbiology

Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A

doi: 10.3389/fmicb.2021.660149

Figure Lengend Snippet: SDS-PAGE analysis of MBP-RNase I and MBP-RNase A fusions. (A) Schematic diagram of MBP-RNase fusion. (B) Total cellular proteins from IPTG-induced cells (NEB Express). (C) Supernatant (soluble) of the fusion proteins (three independent isolates each). Arrows indicate the fusions. (D) Comparison of supernatant (Sol.) and pellet (Incl.) of the fusions. M, protein size marker (NEB). (E) SDS-PAGE analysis of partially purified MBP-RNase I fusion. Lane 1, protein size marker; lane 2, MBP-RNase I fusion purified from IPTG-induced NEB Turbo cells (C2984); lane 3, RNase I f (NEB). (F) RNase activity on fluorescein-labeled RNA (300 nt). No enzyme (uncut) and MBP-ACE2NTD fusion serve as negative controls. S, substrate; P, products.

Article Snippet: RNase I assay: 16 nM of the RNA substrate was digested by purified MBP-RNase I, RNase I (6×His), or refolded RNase I-ACE2NTD (6×His) in a high-salt buffer (NEB buffer 3: 100 mM NaCl, 50 mM Tris–HCl, pH 7.9 at 25°C, 10 mM MgCl 2 , 1 mM DTT) or a high-salt buffer without divalent cations (100 mM NaCl, 50 mM Tris–HCl, pH 7.5, 1 mM DTT) as RNase I is active in the absence of divalent cations or in the presence of EDTA ( ).

Techniques: SDS Page, Marker, Purification, Activity Assay, Labeling

Journal: Frontiers in Microbiology

Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A

doi: 10.3389/fmicb.2021.660149

Figure Lengend Snippet: Purification of RNase I (6×His) and RNase activity assay. (A) Purification of RNase I (6×His) from Nickel-NTA agarose column. Lane 1, RNase I (6×His) pooled fractions from a nickel column (purified from T7 Express cell extract). Arrows indicate the cytoplasmic RNase I precursor (cRNase I) with the signal peptide (predicted MW 30.7 kDa), and the periplasmic RNase I with the signal peptide removed (predicted MW 27.0 kDa). (B) RNase activity on a FAM-labeled SARS-CoV-2 RNA (50 mer). S = substrate; P = cleavage product(s). Positive controls, 50 and 5 U of RNase I f (NEB). RNase I (6×His) enzyme titration (2 μg to 25 ng protein) was used in the activity assay to digest fixed amount of RNA (16 nM) in NEB buffer 3 at 37°C for 1 h. Proteinase K (1.6 U) was added to remove RNase I. The final cleavage products were analyzed by capillary electrophoresis (CE), and peaks were visualized by PeakScan.

Article Snippet: RNase I assay: 16 nM of the RNA substrate was digested by purified MBP-RNase I, RNase I (6×His), or refolded RNase I-ACE2NTD (6×His) in a high-salt buffer (NEB buffer 3: 100 mM NaCl, 50 mM Tris–HCl, pH 7.9 at 25°C, 10 mM MgCl 2 , 1 mM DTT) or a high-salt buffer without divalent cations (100 mM NaCl, 50 mM Tris–HCl, pH 7.5, 1 mM DTT) as RNase I is active in the absence of divalent cations or in the presence of EDTA ( ).

Techniques: Purification, Activity Assay, Nickel Column, Labeling, Titration, Electrophoresis

Journal: Frontiers in Microbiology

Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A

doi: 10.3389/fmicb.2021.660149

Figure Lengend Snippet: SDS-PAGE and Western blot analysis of RNase I-ACE2NTD fusion and activity assays. (A) Schematic diagram of RNase I-ACENTD (6×His) fusion. (B) Western blot analysis of RNase I-ACE2NTD in total protein, supernatant (soluble), and refolded protein using anti-His mAb. (C) Same as in (B) , except using anti-ACE2 mAb. (D) SDS-PAGE analysis of the refolded RNase I-ACE2NTD fusion and further purified protein by Ni magnetic beads and Ni spin column. (E) RNase I-ACE2NTD (refolded) ribonuclease activity on fluorescein (FL)-labeled RNA (300 nt) in NEB buffer 3. RNase I (6×His) and MBP-RNase I were used as positive controls. (F) Ribonuclease activity of RNase I-ACE2NTD (purified by Ni magnetic beads or Ni spin column) on SARS-CoV-2 RNA (50 mer). RNase I f , a positive control. FAM-S, FAM-labeled substrate; FAM-P, FAM labeled cleavage product(s).

Article Snippet: RNase I assay: 16 nM of the RNA substrate was digested by purified MBP-RNase I, RNase I (6×His), or refolded RNase I-ACE2NTD (6×His) in a high-salt buffer (NEB buffer 3: 100 mM NaCl, 50 mM Tris–HCl, pH 7.9 at 25°C, 10 mM MgCl 2 , 1 mM DTT) or a high-salt buffer without divalent cations (100 mM NaCl, 50 mM Tris–HCl, pH 7.5, 1 mM DTT) as RNase I is active in the absence of divalent cations or in the presence of EDTA ( ).

Techniques: SDS Page, Western Blot, Activity Assay, Purification, Magnetic Beads, Labeling, Positive Control

Journal: Frontiers in Microbiology

Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A

doi: 10.3389/fmicb.2021.660149

Figure Lengend Snippet: Escherichia coli expression strains, purification method, and enzyme activity/potential usage.

Article Snippet: RNase I assay: 16 nM of the RNA substrate was digested by purified MBP-RNase I, RNase I (6×His), or refolded RNase I-ACE2NTD (6×His) in a high-salt buffer (NEB buffer 3: 100 mM NaCl, 50 mM Tris–HCl, pH 7.9 at 25°C, 10 mM MgCl 2 , 1 mM DTT) or a high-salt buffer without divalent cations (100 mM NaCl, 50 mM Tris–HCl, pH 7.5, 1 mM DTT) as RNase I is active in the absence of divalent cations or in the presence of EDTA ( ).

Techniques: Expressing, Purification, Activity Assay, Magnetic Beads, Binding Assay, Fluorescence

Journal: Glycobiology

Article Title: Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways

doi: 10.1093/glycob/cwy057

Figure Lengend Snippet: Chromatographic resolution of Siglec-8 ligands extracted from human airway. ( A ) Sephacryl S-500 column chromatography of Siglec-8 ligands extracted from human trachea. Elution of proteins (A 280 , dashed line) and Siglec-8 binding activity (solid line) determined by semiquantitative dot blot Siglec-8-Fc overlay are plotted against the elution volume. ( B ) Composite gel electrophoresis of active binding fractions. Equal aliquots of fractions with Siglec-8-Fc-binding activity detected by semiquantitative dot blot were resolved by 1.5% acrylamide, 2% agarose composite gel electrophoresis, blotted to PVDF membranes and probed for Siglec-8-Fc binding. Images of three replicate gels (boxed) used to accommodate fractions across the active elution fractions were stitched together by lining up common molecular weight standards (Stds, left). Fractions were pooled to represent three size classes of Siglec-8 ligands (designated by estimated molecular weight in daltons): S8-1M, S8-600K and S8-250K.

Article Snippet: Purified Siglec-8-COMP (44 μg) was immobilized on 140 μL of magnetic nickel Sepharose beads (GE Healthcare 28967388).

Techniques: Column Chromatography, Binding Assay, Activity Assay, Dot Blot, Nucleic Acid Electrophoresis, Molecular Weight

Journal: Glycobiology

Article Title: Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways

doi: 10.1093/glycob/cwy057

Figure Lengend Snippet: Siglec-8-COMP affinity purification of Siglec-8 ligands from human trachea. Siglec-8 ligands extracted from human trachea were resolved by Sephacryl S-500 size-exclusion chromatography (Figure ) and fractions corresponding to S8-1M, S8-600K and S8-250K separately combined for affinity purification. Pooled fractions of each molecular size were loaded separately onto 1-mL nickel Sepharose affinity columns carrying 6-His-tagged pentameric Siglec-8-COMP. After loading and washing each column, bound Siglec-8 ligands were eluted with high salt. Equal aliquots of washes and eluates were subjected to 1.5% acrylamide, 2% agarose composite gel electrophoresis, blotted to PVDF membranes and probed with Siglec-8-Fc. Lanes: (1) pooled Sephacryl S-500 fraction; (2) affinity column flow-through; (3) wash 1; (4) wash 2; (5) elution 1; (6) elution 2; (7) elution 3. HiMark-prestained molecular weight standards are shown at the left (Stds).

Article Snippet: Purified Siglec-8-COMP (44 μg) was immobilized on 140 μL of magnetic nickel Sepharose beads (GE Healthcare 28967388).

Techniques: Affinity Purification, Size-exclusion Chromatography, Nucleic Acid Electrophoresis, Affinity Column, Molecular Weight

Journal: Glycobiology

Article Title: Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways

doi: 10.1093/glycob/cwy057

Figure Lengend Snippet: Aggrecan carries Siglec-8-binding glycans. ( A ) Proteins extracted from human trachea were purified by sequential size-exclusion and Siglec-8 affinity chromatography, proteolyzed, and peptides identified by mass spectrometry. Maps of aggrecan protein sequences with identified peptides from three size classes of affinity-purified Siglec-8 ligands are shown. Green bars are peptides (see Table ) that exceed strict false discovery rates for each of the Siglec-8 ligand size classes as indicated. The smaller size classes (S8-600 K and S8-250K) are mapped on aggrecan Uniprot reference sequence P16112 (2415 amino acids); whereas the largest size class (S8-1M) is mapped on C-terminal alternatively spliced aggrecan sequence H0YM81 (2492 amino acids). ( B ) Schematic map of aggrecan. IGD, interglobular domain; CS1, CS2, chondroitin sulfate (CS)-rich domains. Modified from , with permission. ( C ) Co-migration of purified human Siglec-8 ligands and aggrecan immunoreactivity. Three purified size classes of Siglec-8 ligands from human trachea were resolved by electrophoresis on replicate 1.5% acrylamide, 2% agarose composite gels, blotted to PVDF membranes and probed with Siglec-8-Fc, anti-aggrecan antibody 7D4 (α-aggr-G1/2) or anti-aggrecan antibody PA1-1745 (α-aggr-G3). Migration positions of HiMark-prestained standards are indicated at the left. Lanes: (1) S81M; (2) S8-600K and (3) S8-250K. ( D ) Generalized schematic structures of KS and CS chains depicted using symbol nomenclature . Sialic acid and sulfates that are variable are shown in parentheses. ( E ) Elution of S8-1M from Siglec-8-COMP affinity chromatography with soluble glycans. Pooled size-exclusion fractions containing S8-1M (Figure ) were captured on Siglec-8-COMP magnetic beads. The beads were thoroughly washed prior to eluting with β-azidoethylglycosides (lane 1) followed by 500 mM imidazole to elute the bound Siglec-8-COMP with any remaining ligand attached (lane 2). Eluates were resolved by composite gel electrophoresis, blotted and probed with Siglec-8-Fc. Elution was tested with the following β-azidoethylglycosides: none, N -acetyllactosamine (LacNAc, Galβ1-4GlcNAc), 3′-sialyl LacNac (3′Sia-LacNAc, Neu5Acα2-3Galβ1-4GlcNAc), 6′-sulfo-3′-sialyl-LacNAc (6′Su,3′Sia-LacNAc, Neu5Acα2-3[6 S]Galβ1-4GlcNAc) and 6-sulfo-3′-sialyl-LacNAc (6 Su,3′Sia-LacNAc, Neu5Acα2-3Galβ1-4[6S]GlcNAc).

Article Snippet: Purified Siglec-8-COMP (44 μg) was immobilized on 140 μL of magnetic nickel Sepharose beads (GE Healthcare 28967388).

Techniques: Binding Assay, Purification, Affinity Chromatography, Mass Spectrometry, Affinity Purification, Sequencing, Modification, Migration, Electrophoresis, Magnetic Beads, Nucleic Acid Electrophoresis

Journal: Glycobiology

Article Title: Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways

doi: 10.1093/glycob/cwy057

Figure Lengend Snippet: Keratanase and sialidase pretreatments diminish Siglec-8 binding to purified Siglec-8 ligands. Siglec-8 ligands were extracted from human trachea, purified by sequential size-exclusion and Siglec-8 affinity chromatography and subjected to keratanase or sialidase treatments. Samples were resolved on 1.5% acrylamide, 2% agarose composite gels, blotted to PVDF membranes and probed with precomplexed Siglec-8-Fc or anti-aggrecan antibody (7D4) as indicated. Lanes: (1) incubation without enzyme; (2) sialidase (67 mU/mL, 90 min); (3) keratanase II (6 mU/mL, 16 h). HiMark molecular weight standards are shown at the left (Stds).

Article Snippet: Purified Siglec-8-COMP (44 μg) was immobilized on 140 μL of magnetic nickel Sepharose beads (GE Healthcare 28967388).

Techniques: Binding Assay, Purification, Affinity Chromatography, Incubation, Molecular Weight

Journal: Glycobiology

Article Title: Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways

doi: 10.1093/glycob/cwy057

Figure Lengend Snippet: Chondroitinase ABC (ChABC) and keratanase treatments of a human tracheal Siglec-8 ligand reveal a chondroitin sulfate (CS) proteoglycan with Siglec-8-binding keratan sulfate (KS) chains. Siglec-8 ligands were extracted from human trachea and S8-1M purified by sequential size-exclusion and Siglec-8 affinity chromatography. Equal aliquots containing the isolated ligand (S8-1M) or commercial bovine articular cartilage aggrecan (bovine aggrecan) were treated with buffer alone (no enzyme), ChABC, keratanase I or both enzymes for 20 h at 37°C. Samples were denatured and resolved on 1.5% acrylamide, 2% agarose composite gels, blotted to PVDF membranes and probed with precomplexed Siglec-8-Fc overlay or anti-aggrecan antibody. Lanes (all enzyme treatments 20 h at 37°C): (1) no incubation; (2) ChABC (500 mU/mL); (3) keratanase I (21 mU/mL); (4) ChABC (500 mU/mL) plus keratanase I (21 mU/mL); (5) incubation without enzymes. Migration positions of HiMark molecular weight markers are shown.

Article Snippet: Purified Siglec-8-COMP (44 μg) was immobilized on 140 μL of magnetic nickel Sepharose beads (GE Healthcare 28967388).

Techniques: Binding Assay, Purification, Affinity Chromatography, Isolation, Incubation, Migration, Molecular Weight

Journal: Glycobiology

Article Title: Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways

doi: 10.1093/glycob/cwy057

Figure Lengend Snippet: ADAMTS-4 (aggrecanase-1) treatment of human airway Siglec-8 ligands shifts their electrophoretic migration. Siglec-8 ligands were extracted from human trachea, purified by sequential size exclusion followed by Siglec-8 affinity chromatography and subjected to ChABC, aggrecanase or both enzymes. Samples were resolved on 1.5% acrylamide, 2% agarose composite gels, blotted to PVDF membranes and probed with precomplexed Siglec-8-Fc or anti-aggrecan antibody (7D4) as indicated. Lanes (all enzyme treatments 16 h at 37°C): (1) incubation without enzyme; (2) aggrecanase (0.3 mU/mL); (3) aggrecanase (0.3 mU/mL) plus ChABC (0.5 U/mL); (4) ChABC (0.5 U/mL). S8-1M and S8-600K were resolved on 1.5% acrylamide, 2% agarose composite gels with migration positions of HiMark molecular weight markers shown; S8-250K was resolved on 3% acrylamide, 2% agarose composite gels with migration positions of SeeBlue Plus2 molecular weight markers shown.

Article Snippet: Purified Siglec-8-COMP (44 μg) was immobilized on 140 μL of magnetic nickel Sepharose beads (GE Healthcare 28967388).

Techniques: Migration, Purification, Affinity Chromatography, Incubation, Molecular Weight

Journal: Glycobiology

Article Title: Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways

doi: 10.1093/glycob/cwy057

Figure Lengend Snippet: Purified human tracheal Siglec-8 ligand induces human eosinophil apoptosis. Siglec-8 ligands were extracted from human trachea and purified by size-exclusion chromatography followed by Siglec-8 affinity chromatography. A portion of purified S8-250K was treated with cold periodate to selectively oxidize the glycerol sidearm of its sialic acid followed by sodium borohydride reduction (negative control). An equal portion was incubated without periodate and treated with sodium borohydride (intact ligand). Oxidized and intact S8-250K were dialyzed against RPMI medium and added at equal concentrations to freshly isolated primary human eosinophils. After 24 h in culture, eosinophil apoptosis was quantified by flow cytometry. ( A ) Oxidized and intact S8-250 K were electrophoretically resolved on a composite agarose–acrylamide gel, blotted to PVDF membranes. Replicate blots were probed with Siglec-8-Fc to reveal Siglec-8 ligands or anti-aggrecan antibody (7D4). Lanes: (1) untreated S8-250K; (2) periodate oxidized and reduced S8-250K; (3) reduced S8-250K (intact ligand). ( B ) After isolation and overnight interleukin 5 (IL-5) priming, human eosinophils were incubated with equal portions of oxidized and intact S8-250K and apoptosis was assessed 18–24 h later. Results are expressed relative to untreated eosinophils, which had average apoptosis of 42 ± 5% (SEM). Data are displayed as mean and SEM of six replicates performed on three separate primary human eosinophil preparations.

Article Snippet: Purified Siglec-8-COMP (44 μg) was immobilized on 140 μL of magnetic nickel Sepharose beads (GE Healthcare 28967388).

Techniques: Purification, Size-exclusion Chromatography, Affinity Chromatography, Negative Control, Incubation, Isolation, Flow Cytometry, Acrylamide Gel Assay

Journal: Frontiers in microbiology

Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A.

doi: 10.3389/fmicb.2021.660149

Figure Lengend Snippet: FIGURE 3 | Purification of RNase I (6×His) and RNase activity assay. (A) Purification of RNase I (6×His) from Nickel-NTA agarose column. Lane 1, RNase I (6×His) pooled fractions from a nickel column (purified from T7 Express cell extract). Arrows indicate the cytoplasmic RNase I precursor (cRNase I) with the signal peptide (predicted MW 30.7 kDa), and the periplasmic RNase I with the signal peptide removed (predicted MW 27.0 kDa). (B) RNase activity on a FAM-labeled SARS-CoV-2 RNA (50 mer). S = substrate; P = cleavage product(s). Positive controls, 50 and 5 U of RNase If (NEB). RNase I (6×His) enzyme titration (2 µg to 25 ng protein) was used in the activity assay to digest fixed amount of RNA (16 nM) in NEB buffer 3 at 37◦C for 1 h. Proteinase K (1.6 U) was added to remove RNase I. The final cleavage products were analyzed by capillary electrophoresis (CE), and peaks were visualized by PeakScan.

Article Snippet: To reduce protein purification cost, expression of RNase I-ACE2 (6×His) and hRNase A-ACE2 (6×His) fusions may be carried out using the K. lactis yeast expression system (NEB) to export the recombinant proteins into the culture medium for industrial production of the fusions.

Techniques: Activity Assay, Nickel Column, Labeling, Titration, Electrophoresis

Journal: Frontiers in microbiology

Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A.

doi: 10.3389/fmicb.2021.660149

Figure Lengend Snippet: FIGURE 5 | SDS-PAGE and Western blot analysis of RNase I-ACE2NTD fusion and activity assays. (A) Schematic diagram of RNase I-ACENTD (6×His) fusion. (B) Western blot analysis of RNase I-ACE2NTD in total protein, supernatant (soluble), and refolded protein using anti-His mAb. (C) Same as in (B), except using anti-ACE2 mAb. (D) SDS-PAGE analysis of the refolded RNase I-ACE2NTD fusion and further purified protein by Ni magnetic beads and Ni spin column. (E) RNase I-ACE2NTD (refolded) ribonuclease activity on fluorescein (FL)-labeled RNA (300 nt) in NEB buffer 3. RNase I (6×His) and MBP-RNase I were used as positive controls. (F) Ribonuclease activity of RNase I-ACE2NTD (purified by Ni magnetic beads or Ni spin column) on SARS-CoV-2 RNA (50 mer). RNase If, a positive control. FAM-S, FAM-labeled substrate; FAM-P, FAM labeled cleavage product(s).

Article Snippet: To reduce protein purification cost, expression of RNase I-ACE2 (6×His) and hRNase A-ACE2 (6×His) fusions may be carried out using the K. lactis yeast expression system (NEB) to export the recombinant proteins into the culture medium for industrial production of the fusions.

Techniques: SDS Page, Western Blot, Activity Assay, Magnetic Beads, Labeling, Positive Control

Journal: Frontiers in microbiology

Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A.

doi: 10.3389/fmicb.2021.660149

Figure Lengend Snippet: FIGURE 7 | SDS-PAGE analysis of purified hRNase A-ACE2NTD150 (6×His) and ribonuclease activity assays. (A) Partially purified hRNase A-ACE2NTD150 (6×His) (no signal peptide) by Ni-NTA agarose column chromatography or by binding to Ni magnetic beads. (B) RNase activity on FAM-labeled SARS-CoV-2 RNA (50 mer). Arrows indicate the substrate and cleavage products in the CE assay. (C) RNase activity assay on a 300-nt RNA and low MW RNA ladder as analyzed on a 6% PAG-urea gel (stained with SYBR green and visualized on a Typhoon Imager).

Article Snippet: To reduce protein purification cost, expression of RNase I-ACE2 (6×His) and hRNase A-ACE2 (6×His) fusions may be carried out using the K. lactis yeast expression system (NEB) to export the recombinant proteins into the culture medium for industrial production of the fusions.

Techniques: SDS Page, Activity Assay, Column Chromatography, Binding Assay, Magnetic Beads, Labeling, Staining, SYBR Green Assay