An efficient system to generate truncated human angiotensin converting enzyme 2 (hACE2) capable of binding RBD and spike protein of SARS-CoV2

Protein Expr Purif. 2021 Aug:184:105889. doi: 10.1016/j.pep.2021.105889. Epub 2021 Apr 11.

Abstract

Human angiotensin converting enzyme 2 (hACE2) mediates the cell entry of both SARS-CoV and SARS-CoV2 and can be used as a drug target. The DNA encoding the truncated hACE2 (30-356aa) was cloned into pET-28a (+) and expressed in Escherichia coli Rosetta (DE3). The recombinant hACE2 (rhACE2) was purified by affinity chromatography on a Ni-NTA column and characterized with SDS-PAGE and Western blot. The binding activity of rhACE2 to Spike protein of SARS-CoV2 was evaluated in S protein-overexpressed HEK293A cells (HEK293A-SP cells) through flow cytometry. The prokaryotic expression system is able to produce approximately 75 mg protein per liter, which would be useful for infection mechanism study, and drug screening and development of SARS-CoV2.

Keywords: COVID-19; RBD; SARS-CoV2; Spike protein; hACE2.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Angiotensin-Converting Enzyme 2* / chemistry
  • Angiotensin-Converting Enzyme 2* / genetics
  • Angiotensin-Converting Enzyme 2* / isolation & purification
  • Angiotensin-Converting Enzyme 2* / metabolism
  • COVID-19 / virology
  • Chromatography, Affinity
  • Cloning, Molecular
  • Escherichia coli / genetics
  • HEK293 Cells
  • Humans
  • Protein Binding
  • Protein Domains
  • Recombinant Proteins* / chemistry
  • Recombinant Proteins* / genetics
  • Recombinant Proteins* / isolation & purification
  • Recombinant Proteins* / metabolism
  • Spike Glycoprotein, Coronavirus / metabolism*

Substances

  • Recombinant Proteins
  • Spike Glycoprotein, Coronavirus
  • spike protein, SARS-CoV-2
  • ACE2 protein, human
  • Angiotensin-Converting Enzyme 2