title: Coxsackievirus B3 protease 3C: expression, purification, crystallization and preliminary structural insights.
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 MgCl2in 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.
authors.affiliation: Section of Genetics, Cell Biology and Development, Department of Biology, University of Patras, University Campus, 26500 Patras, Greece.
authors.forename: Stavroula
authors.initials: S
authors.lastname: Fili
authors.affiliation: Section of Genetics, Cell Biology and Development, Department of Biology, University of Patras, University Campus, 26500 Patras, Greece.
authors.forename: Alexandros
authors.initials: A
authors.lastname: Valmas
authors.affiliation: Section of Genetics, Cell Biology and Development, Department of Biology, University of Patras, University Campus, 26500 Patras, Greece.
authors.forename: Magdalini
authors.initials: M
authors.lastname: Christopoulou
authors.affiliation: Section of Genetics, Cell Biology and Development, Department of Biology, University of Patras, University Campus, 26500 Patras, Greece.
authors.forename: Maria
authors.initials: M
authors.lastname: Spiliopoulou
authors.affiliation: Section of Genetics, Cell Biology and Development, Department of Biology, University of Patras, University Campus, 26500 Patras, Greece.
authors.forename: Nikos
authors.initials: N
authors.lastname: Nikolopoulos
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, 13288 Marseille, France.
authors.forename: Julie
authors.initials: J
authors.lastname: Lichière
authors.affiliation: Section of Genetics, Cell Biology and Development, Department of Biology, University of Patras, University Campus, 26500 Patras, Greece.
authors.forename: Souzana
authors.initials: S
authors.lastname: Logotheti
authors.affiliation: Section of Genetics, Cell Biology and Development, Department of Biology, University of Patras, University Campus, 26500 Patras, Greece.
authors.forename: Fotini
authors.initials: F
authors.lastname: Karavassili
authors.affiliation: Section of Genetics, Cell Biology and Development, Department of Biology, University of Patras, University Campus, 26500 Patras, Greece.
authors.forename: Eleftheria
authors.initials: E
authors.lastname: Rosmaraki
authors.affiliation: European Synchrotron Radiation Facility, BP 220, 38043 Grenoble CEDEX 9, France.
authors.forename: Andrew
authors.initials: A
authors.lastname: Fitch
authors.affiliation: European Synchrotron Radiation Facility, BP 220, 38043 Grenoble CEDEX 9, France.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Wright
authors.affiliation: PANalytical B.V., Lelyweg 1, 7602 EA Almelo, The Netherlands.
authors.forename: Detlef
authors.initials: D
authors.lastname: Beckers
authors.affiliation: PANalytical B.V., Lelyweg 1, 7602 EA Almelo, The Netherlands.
authors.forename: Thomas
authors.initials: T
authors.lastname: Degen
authors.affiliation: PANalytical B.V., Lelyweg 1, 7602 EA Almelo, The Netherlands.
authors.forename: Gwilherm
authors.initials: G
authors.lastname: Nénert
authors.affiliation: Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, 23562 Lübeck, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Hilgenfeld
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, 13288 Marseille, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Papageorgiou
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, 13288 Marseille, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Canard
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, 13288 Marseille, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Coutard
authors.affiliation: Section of Genetics, Cell Biology and Development, Department of Biology, University of Patras, University Campus, 26500 Patras, Greece.
authors.forename: Irene
authors.initials: I
authors.lastname: Margiolaki
mesh_terms: D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029822:Enterovirus B, Human / Q000737:chemistry* / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014961:X-Ray Diffraction
chemical_list: D011994:Recombinant Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases
keywords: 3C protease; coxsackievirus B3; powder diffraction