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
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Abstract
The SARS-CoV-2 viral genome contains a positive-strand single-stranded RNA of ~30 kb. Human ACE2 protein is the receptor for SARS-CoV-2 virus attachment and initiation of infection. We propose to use ribonucleases (RNases) as antiviral agents to destroy the viral genome in vitro. In the virions the RNA is protected by viral capsid proteins, membrane proteins and nucleocapsid proteins. To overcome this protection we set out to construct RNase fusion with human ACE2 receptor N-terminal domain (ACE2NTD). We constructed six proteins expressed in E. coli cells: 1) MBP-ACE2NTD, 2) ACE2NTD-GFP, 3) RNase I (6xHis), 4) RNase III (6xHis), 5) RNase I-ACE2NTD (6xHis), and 6) human RNase A-ACE2NTD150 (6xHis). We evaluated fusion expression in different E. coli strains, partially purified MBP-ACE2NTD protein from the soluble fraction of bacterial cell lysate, and refolded MBP-ACE2NTD protein from inclusion body. The engineered RNase I-ACE2NTD (6xHis) and hRNase A-ACE2NTD (6xHis) fusions are active in cleaving COVID-19 RNA in vitro. The recombinant RNase I (6xHis) and RNase III (6xHis) are active in cleaving RNA and dsRNA in test tube. This study provides a proof-of-concept for construction of fusion protein between human cell receptor and nuclease that may be used to degrade viral nucleic acids in our environment.
Graphical Abstract
Cartoon illustration part of this work (Human ACE2 N-terminal domain tethered to RNase A and RNA degradation by the fusion enzyme).
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SciScore for 10.1101/2021.01.31.429007: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Antibodies Sentences Resources Protein pull-down assays (protein complex pull-down by Ni magnetic beads): Western blot using anti-6xHis antibody (Ab) and mouse monoclonal anti-ACE2 Ab: Mouse monoclonal anti-ACE2 Ab and mouse anti-6xHis Ab were purchased from Cell Signaling Technologies (CST, MA) and EMC Millipore company, respectively anti-6xHissuggested: Noneanti-ACE2suggested: NoneThe excess primary antibodies were washed off with 1x PBS containing 0.1% Tween 20 and 0.1% Triton X100 three times for 15 min, followed by … SciScore for 10.1101/2021.01.31.429007: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Antibodies Sentences Resources Protein pull-down assays (protein complex pull-down by Ni magnetic beads): Western blot using anti-6xHis antibody (Ab) and mouse monoclonal anti-ACE2 Ab: Mouse monoclonal anti-ACE2 Ab and mouse anti-6xHis Ab were purchased from Cell Signaling Technologies (CST, MA) and EMC Millipore company, respectively anti-6xHissuggested: Noneanti-ACE2suggested: NoneThe excess primary antibodies were washed off with 1x PBS containing 0.1% Tween 20 and 0.1% Triton X100 three times for 15 min, followed by incubation with secondary anti-mouse IgG HPR conjugated antibodies (1:1000) (CST) and secondary Ab, washed three times with 1x PBS, 0.1% Tween20 and 0.1% Triton X100. anti-mouse IgG HPRsuggested: NoneResults from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from scite Reference Check: We found citations with errata. We recommend checking the errata to confirm that they do not impact the accuracy of your citation.
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