Molecular cloning, overproduction, purification and biochemical characterization of the p39 nsp2 protease domains encoded by three alphaviruses

Di Zhang, J. Tőzsér, David S. Waugh

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Alphaviruses cause serious diseases that pose a potential health threat to both humans and livestock. The nonstructural protein 2 (nsp2) encoded by alphaviruses is a multifunctional enzyme that is essential for viral replication and maturation. Its 39-kDa C-terminal domain (nsp2pro) is a cysteine protease that is responsible for cleaving a viral polyprotein at three sites to generate nonstructural proteins 1, 2, 3 and 4. In the present study, we evaluated nsp2pro domains from the following three sources as reagents for site-specific cleavage of fusion proteins: Venezuelan Equine Encephalitis Virus (VEEV), Semliki Forest Virus (SFV) and Sindbis Virus (SIN). All three alphavirus proteases cleaved model fusion protein substrates with high specificity but they were much less efficient enzymes than potyviral proteases from tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV). Oligopeptide substrates were also cleaved with very low efficiency by the alphavirus proteases. We conclude that, in general, alphavirus nsp2pro proteases are not very useful tools for the removal of affinity tags from recombinant proteins although they do remain promising therapeutic targets for the treatment of a variety of diseases.

Original languageEnglish
Pages (from-to)89-97
Number of pages9
JournalProtein Expression and Purification
Volume64
Issue number1
DOIs
Publication statusPublished - Mar 2009

Fingerprint

Alphavirus
Molecular Cloning
Peptide Hydrolases
Proteins
Venezuelan Equine Encephalitis Viruses
Multifunctional Enzymes
Semliki forest virus
Sindbis Virus
Polyproteins
Oligopeptides
Cysteine Proteases
Livestock
Recombinant Proteins
Tobacco
Veins
Viruses
Health
Enzymes

Keywords

  • Affinity tag
  • Fusion protein
  • Maltose-binding protein
  • Nonstructural protein 2
  • Protease
  • Semliki forest virus
  • Sindbis virus
  • Venezuelan equine encephalitis virus

ASJC Scopus subject areas

  • Biotechnology

Cite this

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abstract = "Alphaviruses cause serious diseases that pose a potential health threat to both humans and livestock. The nonstructural protein 2 (nsp2) encoded by alphaviruses is a multifunctional enzyme that is essential for viral replication and maturation. Its 39-kDa C-terminal domain (nsp2pro) is a cysteine protease that is responsible for cleaving a viral polyprotein at three sites to generate nonstructural proteins 1, 2, 3 and 4. In the present study, we evaluated nsp2pro domains from the following three sources as reagents for site-specific cleavage of fusion proteins: Venezuelan Equine Encephalitis Virus (VEEV), Semliki Forest Virus (SFV) and Sindbis Virus (SIN). All three alphavirus proteases cleaved model fusion protein substrates with high specificity but they were much less efficient enzymes than potyviral proteases from tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV). Oligopeptide substrates were also cleaved with very low efficiency by the alphavirus proteases. We conclude that, in general, alphavirus nsp2pro proteases are not very useful tools for the removal of affinity tags from recombinant proteins although they do remain promising therapeutic targets for the treatment of a variety of diseases.",
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