Coxsackievirus B3 protease 3C: expression, purification, crystallization and preliminary structural insights

Acta Crystallogr F Struct Biol Commun. 2016 Dec 1;72(Pt 12):877-884. doi: 10.1107/S2053230X16018513. Epub 2016 Nov 25.

Abstract

Viral proteases are proteolytic enzymes that orchestrate the assembly of viral components during the viral life cycle and proliferation. Here, the expression, purification, crystallization and preliminary X-ray diffraction analysis are presented of protease 3C, the main protease of an emerging enterovirus, coxsackievirus B3, that is responsible for many cases of viral myocarditis. Polycrystalline protein precipitates suitable for X-ray powder diffraction (XRPD) measurements were produced in the presence of 22-28%(w/v) PEG 4000, 0.1 M Tris-HCl, 0.2 M MgCl2 in a pH range from 7.0 to 8.5. A polymorph of monoclinic symmetry (space group C2, unit-cell parameters a = 77.9, b = 65.7, c = 40.6 Å, β = 115.9°) was identified via XRPD. These results are the first step towards the complete structural determination of the molecule via XRPD and a parallel demonstration of the accuracy of the method.

Keywords: 3C protease; coxsackievirus B3; powder diffraction.

MeSH terms

  • Amino Acid Sequence
  • Cloning, Molecular
  • Crystallization
  • Crystallography, X-Ray
  • Cysteine Endopeptidases / chemistry*
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / metabolism
  • Enterovirus B, Human / chemistry*
  • Enterovirus B, Human / enzymology
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Plasmids / chemistry
  • Plasmids / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Viral Proteins / chemistry*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism
  • X-Ray Diffraction

Substances

  • Recombinant Proteins
  • Viral Proteins
  • Cysteine Endopeptidases
  • 3C proteases