Journal: bioRxiv

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: 10.1101/2021.01.31.429007

Figure Lengend Snippet: A. A schematic diagram of MBP-RNase A fusion. B. Total cellular proteins from IPTG-induced cells (NEB Express). C. Supernatant (soluble) of the fusion proteins (3 independent isolates each). Arrows indicate the fusions. D. Comparison of supernatant (Sol.) and pellet (Inc.) 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; 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.

Article Snippet: RNase I assay: 16 nM of the RNA substrate was digested by purified MBP-RNase I, RNase I (6xHis) or refolded RNase I-ACE2NTD (6xHis) 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: Marker, SDS Page, Purification, Activity Assay, Labeling

Journal: bioRxiv

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: 10.1101/2021.01.31.429007

Figure Lengend Snippet: A. Purification of RNase I (6xHis) from Nickel-NTA agarose column. Lane 1, RNase I (6xHis) 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 COVID-19 RNA (60 mer). S = substrate; P = cleavage product(s). Positive controls, 50 and 5 U of RNase I f (MBP-RNase I fusion, NEB). RNase I (6xHis) 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 (6xHis) or refolded RNase I-ACE2NTD (6xHis) 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, Nickel Column, Activity Assay, Labeling, Titration, Electrophoresis

Journal: bioRxiv

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: 10.1101/2021.01.31.429007

Figure Lengend Snippet: A. Schematic diagram of RNase I-ACENTD (6xHis) fusion. B. Western blot analysis of RNase I-ACE2NTD in total protein, supernatant (soluble), and refolded protein using anti-6xHis Ab. C. Same as in B , except using anti-ACE2 monoclonal Ab. 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 DNA (300 nt) in NEB buffer 3. RNase I (6xHis) 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 COVID-19 RNA (60mer). 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 (6xHis) or refolded RNase I-ACE2NTD (6xHis) 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: Western Blot, SDS Page, Purification, Magnetic Beads, Activity Assay, Labeling, Positive Control

Journal: bioRxiv

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: 10.1101/2021.01.31.429007

Figure Lengend Snippet: A. Partially purified hRNase A-ACE2NTD150 (6xHis) (no signal peptide) by Ni-NTA agarose column chromatography or by binding to Ni magnetic beads. B. RNase activity on FAM-labeled COVID-19 RNA (60mer). 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: RNase I assay: 16 nM of the RNA substrate was digested by purified MBP-RNase I, RNase I (6xHis) or refolded RNase I-ACE2NTD (6xHis) 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, Column Chromatography, Binding Assay, Magnetic Beads, Activity Assay, Labeling, Staining, SYBR Green Assay