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 1 | SDS-PAGE analysis of maltose binding protein-angiotensin converting enzyme 2 N-terminal domain (MBP-ACE2NTD) fusion. (A) A schematic diagram of the fusion protein. (B) Total cellular proteins from IPTG-induced cells (NEB Express) containing the MBP-ACE2NTD fusion. Nine out of 11 clones tested here expressed the fusion (except #1 and #4). Clone #8 was chosen for further study. (C) MBP-ACE2NTD fusion expressed from IPTG induced cells (T7 Shuffle, lanes 1–4). Lane 1, total cellular protein; lane 2, flow-through from an amylose column; lane 3, eluted MBP-ACE2NTD protein from an amylose column; lane 4, MBP-ACE2NTD protein found in the pellet (inclusion body); lane 5, refolded MBP-ACE2NTD fusion protein from cell pellet (NEB Express); M, protein size marker (10–200 kDa, NEB). Arrows indicate the MBP-ACE2NTD protein, a truncated protein, and host MBP. (D) Purified MBP-ACE2NTD protein by amylose magnetic beads. Lane 1, refolded fusion; lanes 2 and 3, protein eluted in a Tris–HCl buffer (20 mM, pH 7.5) and sodium phosphate buffer (0.1 M, pH 8.0) with 10 mM maltose.

Article Snippet: Ab Mouse monoclonal anti-ACE2 Ab (catalog number #74512) and mouse anti-His tag Ab (catalog number 70796-3) were purchased from Cell Signaling Technologies (CST, MA) and EMC Millipore (Germany), respectively.

Techniques: SDS Page, Binding Assay, Clone Assay, Marker, Magnetic Beads

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: Ab Mouse monoclonal anti-ACE2 Ab (catalog number #74512) and mouse anti-His tag Ab (catalog number 70796-3) were purchased from Cell Signaling Technologies (CST, MA) and EMC Millipore (Germany), respectively.

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 6 | Expression of hRNase A-ACE2NTD150 in T7 Express LysY/lacIq (C3013). (A) Schematic diagram of the fusion protein. (B) SDS-PAGE analysis of five isolates of hRNase A-ACE2NTD150 (five isolates with MBP signal peptide, five clones without MBP signal peptide). Total cell lysate of pET21b serves as a negative control. (C,D) Western blot analysis of the fusion proteins (soluble fraction/supernatant) by anti-His mAb and anti-ACE2 mAb.

Article Snippet: Ab Mouse monoclonal anti-ACE2 Ab (catalog number #74512) and mouse anti-His tag Ab (catalog number 70796-3) were purchased from Cell Signaling Technologies (CST, MA) and EMC Millipore (Germany), respectively.

Techniques: Expressing, SDS Page, Clone Assay, Negative Control, Western Blot