Molecular cloning, expression, purification, and mass spectrometric characterization of 3C-like protease of SARS coronavirus

Protein Expr Purif. 2003 Dec;32(2):302-8. doi: 10.1016/j.pep.2003.08.016.

Abstract

Severe acute respiratory syndrome (SARS) is an acute respiratory illness, which has broken out in China. It has been known that SARS coronavirus (SARS_CoV) is a novel human coronavirus and is responsible for SARS infection. Belonging to one of the major proteins associated with SARS_CoV, SARS 3C-like protease (SARS_3CL(pro)) functions as a cysteine protease engaging in the proteolytic cleavage of the viral precursor polyprotein to a series of functional proteins required for coronavirus replication and is considered as an appealing target for designing anti-SARS agents. To facilitate the studies regarding the functions and structures of SARS_3CL(pro), in this report the synthetic genes encoding 3CL(pro) of SARS_CoV were assembled, and the plasmid was constructed using pQE30 as vector and expressed in Escherichia coli M15 cells. The highly yielded ( approximately 15mg/L) expressed protease was purified by use of NTA-Ni(2+) affinity chromatography and FPLC system, and its sequence was determined by LC/MS with the residue coverage of 46.4%.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Chromatography, Affinity
  • Chromatography, Gel
  • Cloning, Molecular
  • Coronavirus 3C Proteases
  • Cysteine Endopeptidases
  • Electrophoresis, Polyacrylamide Gel
  • Endopeptidases / biosynthesis*
  • Endopeptidases / chemistry*
  • Endopeptidases / genetics
  • Endopeptidases / isolation & purification
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Molecular Sequence Data
  • Nickel / chemistry
  • Nitrilotriacetic Acid / chemistry
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Spectrometry, Mass, Electrospray Ionization / methods
  • Viral Proteins / biosynthesis*
  • Viral Proteins / chemistry*
  • Viral Proteins / genetics
  • Viral Proteins / isolation & purification

Substances

  • Recombinant Proteins
  • Viral Proteins
  • Nickel
  • Endopeptidases
  • Cysteine Endopeptidases
  • Coronavirus 3C Proteases
  • Nitrilotriacetic Acid