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
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 infection. We propose to use ribonucleases (RNases) as antiviral agents to destroy the viral genome in vitro. In the virions, the RNA is protec...
Main Authors: | , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
Frontiers Media S.A.
2021-06-01
|
Series: | Frontiers in Microbiology |
Subjects: | |
Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2021.660149/full |
id |
doaj-a49ee93ac12144d0b2b782c712df862c |
---|---|
record_format |
Article |
spelling |
doaj-a49ee93ac12144d0b2b782c712df862c2021-06-11T11:00:03ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2021-06-011210.3389/fmicb.2021.660149660149Expression 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 AShuang-yong XuAlexey FomenkovTien-Hao ChenErbay YigitThe 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 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 utilize RNases as antiviral strategy, we set out to construct RNase fusion with human ACE2 receptor N-terminal domain (ACE2NTD). We expressed six proteins in E. coli cells: (1) MBP-ACE2NTD, (2) ACE2NTD-GFP, (3) RNase I (6×His), (4) RNase III (6×His), (5) RNase I-ACE2NTD (6×His), and (6) human RNase A-ACE2NTD (6×His). 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 (6×His) and hRNase A-ACE2NTD (6×His) fusions are active in cleaving SARS-CoV-2 RNA fragment in vitro. The recombinant RNase I (6×His) and RNase III (6×His) are active in cleaving RNA and dsRNA in test tube. This study provides a proof-of-concept for construction of fusion protein between human receptor and nuclease that may be used to degrade viral nucleic acids.https://www.frontiersin.org/articles/10.3389/fmicb.2021.660149/fullE. coli ribonuclease I (RNase I)RNase IIIRNase I-ACE2NTD fusionhuman RNase A-ACE2NTD fusionSARS-CoV-2human ACE2 receptor |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Shuang-yong Xu Alexey Fomenkov Tien-Hao Chen Erbay Yigit |
spellingShingle |
Shuang-yong Xu Alexey Fomenkov Tien-Hao Chen Erbay Yigit 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 Frontiers in Microbiology E. coli ribonuclease I (RNase I) RNase III RNase I-ACE2NTD fusion human RNase A-ACE2NTD fusion SARS-CoV-2 human ACE2 receptor |
author_facet |
Shuang-yong Xu Alexey Fomenkov Tien-Hao Chen Erbay Yigit |
author_sort |
Shuang-yong Xu |
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 |
title_short |
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 |
title_full |
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 |
title_fullStr |
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 |
title_full_unstemmed |
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 |
title_sort |
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 |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Microbiology |
issn |
1664-302X |
publishDate |
2021-06-01 |
description |
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 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 utilize RNases as antiviral strategy, we set out to construct RNase fusion with human ACE2 receptor N-terminal domain (ACE2NTD). We expressed six proteins in E. coli cells: (1) MBP-ACE2NTD, (2) ACE2NTD-GFP, (3) RNase I (6×His), (4) RNase III (6×His), (5) RNase I-ACE2NTD (6×His), and (6) human RNase A-ACE2NTD (6×His). 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 (6×His) and hRNase A-ACE2NTD (6×His) fusions are active in cleaving SARS-CoV-2 RNA fragment in vitro. The recombinant RNase I (6×His) and RNase III (6×His) are active in cleaving RNA and dsRNA in test tube. This study provides a proof-of-concept for construction of fusion protein between human receptor and nuclease that may be used to degrade viral nucleic acids. |
topic |
E. coli ribonuclease I (RNase I) RNase III RNase I-ACE2NTD fusion human RNase A-ACE2NTD fusion SARS-CoV-2 human ACE2 receptor |
url |
https://www.frontiersin.org/articles/10.3389/fmicb.2021.660149/full |
work_keys_str_mv |
AT shuangyongxu expressionofhumanace2nterminaldomainpartofthereceptorforsarscov2infusionwithmaltosebindingproteinecoliribonucleaseiandhumanrnasea AT alexeyfomenkov expressionofhumanace2nterminaldomainpartofthereceptorforsarscov2infusionwithmaltosebindingproteinecoliribonucleaseiandhumanrnasea AT tienhaochen expressionofhumanace2nterminaldomainpartofthereceptorforsarscov2infusionwithmaltosebindingproteinecoliribonucleaseiandhumanrnasea AT erbayyigit expressionofhumanace2nterminaldomainpartofthereceptorforsarscov2infusionwithmaltosebindingproteinecoliribonucleaseiandhumanrnasea |
_version_ |
1721382453907554304 |