title: Improvement of the purification of Saint Louis encephalitis virus NS2B-NS3 recombinant protease expressed in Escherichia coli.
abstract: The NS2B-NS3 serine protease of Saint Louis Encephalitis virus (SLEV), a potential target for antiviral drug design, has been over-expressed as a recombinant His-tag protein in Escherichia coli for future structural determination. The production process resulted in a soluble protease with co-purification of DnaK, a bacterial molecular chaperone already described in E. coli protein expression. Two approaches were tested to remove this specific contaminant. The fusion protein bound to the purification resin was washed with MgATP plus soluble denatured E. coli proteins before elution, but this method proved to be poorly efficient due to a substantial loss of the targeted recombinant protease. After the immobilized metal affinity chromatography step, the use of gel permeation chromatography with addition of arginine in the mobile phase led to effective separation of the native viral protease from the DnaK aggregates. By this way, SLEV DeltaNS2B-NS3pro protease was purified as a functional protein with a purity greater than 90% suitable for crystallization attempts.
authors.affiliation: Unité de Virologie Tropicale, Institut de Médecine Tropicale du Service de Santé des Armées (IMTSSA), BP 46, Marseille Armées, France. publi.viro@laposte.net
authors.forename: Boris
authors.initials: B
authors.lastname: Pastorino
authors.forename: Dominique
authors.initials: D
authors.lastname: Rolland
authors.forename: Christophe N
authors.initials: CN
authors.lastname: Peyrefitte
authors.forename: Nathalie
authors.initials: N
authors.lastname: Wurtz
authors.forename: Lionel
authors.initials: L
authors.lastname: Almeras
authors.forename: Maël
authors.initials: M
authors.lastname: Bessaud
authors.forename: Hugues J
authors.initials: HJ
authors.lastname: Tolou
mesh_terms: D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004665:Encephalitis Virus, St. Louis / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D020365:RNA Helicases / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000235:genetics / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000302:isolation & purification*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D020365:RNA Helicases; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C080102:NS2B protein, flavivirus; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases