Expression and purification of SARS coronavirus proteins using SUMO-fusions

Protein Expr Purif. 2005 Jul;42(1):100-10. doi: 10.1016/j.pep.2005.02.004. Epub 2005 Feb 23.

Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV) proteins belong to a large group of proteins that is difficult to express in traditional expression systems. The ability to express and purify SARS-CoV proteins in large quantities is critical for basic research and for development of pharmaceutical agents. The work reported here demonstrates: (1) fusion of SUMO (small ubiquitin-related modifier), a 100 amino acid polypeptide, to the N-termini of SARS-CoV proteins dramatically enhances expression in Escherichia coli cells and (2) 6x His-tagged SUMO-fusions facilitate rapid purification of the viral proteins on a large scale. We have exploited the natural chaperoning properties of SUMO to develop an expression system suitable for proteins that cannot be expressed by traditional methodologies. A unique feature of the system is the SUMO tag, which enhances expression, facilitates purification, and can be efficiently cleaved by a SUMO-specific protease to generate native protein with a desired N-terminus. We have purified various SARS-CoV proteins under either native or denaturing conditions. These purified proteins have been used to generate highly specific polyclonal antibodies. Our study suggests that the SUMO-fusion technology will be useful for enhancing expression and purification of the viral proteins for structural and functional studies as well as for therapeutic uses.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Coronavirus 3C Proteases
  • Coronavirus Nucleocapsid Proteins
  • Cysteine Endopeptidases / biosynthesis
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / isolation & purification
  • Escherichia coli / genetics
  • Gene Expression / genetics*
  • Genetic Vectors / genetics
  • Histidine / genetics
  • Membrane Glycoproteins / biosynthesis
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / isolation & purification
  • Nucleocapsid Proteins / biosynthesis
  • Nucleocapsid Proteins / genetics
  • Nucleocapsid Proteins / isolation & purification
  • Peptide Hydrolases / metabolism
  • Recombinant Fusion Proteins / biosynthesis*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • SARS Virus / genetics*
  • Small Ubiquitin-Related Modifier Proteins / genetics*
  • Spike Glycoprotein, Coronavirus
  • Viral Envelope Proteins / biosynthesis
  • Viral Envelope Proteins / genetics
  • Viral Envelope Proteins / isolation & purification
  • Viral Proteins / genetics*
  • Viral Proteins / isolation & purification
  • Viral Proteins / metabolism

Substances

  • Coronavirus Nucleocapsid Proteins
  • Membrane Glycoproteins
  • Nucleocapsid Proteins
  • Recombinant Fusion Proteins
  • Small Ubiquitin-Related Modifier Proteins
  • Spike Glycoprotein, Coronavirus
  • Viral Envelope Proteins
  • Viral Proteins
  • spike glycoprotein, SARS-CoV
  • polyhistidine
  • Histidine
  • Peptide Hydrolases
  • Cysteine Endopeptidases
  • Coronavirus 3C Proteases