title: Improved purification protocol for wild-type and mutant human foamy virus proteases.
abstract: Wild-type and an active site mutant (S25T) human foamy virus (HFV) proteases were expressed in fusion with maltose binding protein in Escherichia coli. The mutant enzyme contained a Ser to Thr mutation in the -Asp-Ser-Gly- active site triplet of the enzyme, which forms the "fireman's grip" between the two subunits of the homodimeric enzyme. The fusion proteins were purified by affinity chromatography on amylose resin, cleaved with factor Xa, and the processed enzymes were purified by gel filtration under denaturing condition. Refolding after purification resulted in active enzymes with comparable yields. Furthermore, both enzymes showed similar catalytic activities in an oligopeptide substrate representing an HFV Gag cleavage site. However, the S25T mutant showed increased stability in urea unfolding experiment, in a good agreement with the suggested role of the Thr residue of fireman's grip.
authors.affiliation: Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, H-4012 Debrecen, Nagyerdei krt. 98., P.O. Box 6, Hungary.
authors.forename: Péter
authors.initials: P
authors.lastname: Boross
authors.forename: József
authors.initials: J
authors.lastname: Tözsér
authors.forename: Péter
authors.initials: P
authors.lastname: Bagossi
mesh_terms: D019943:Amino Acid Substitution; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001665:Binding Sites / Q000235:genetics; D002846:Chromatography, Affinity / Q000379:methods; D019281:Dimerization; D004926:Escherichia coli; D006801:Humans; D017354:Point Mutation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D016282:Aspartic Acid Endopeptidases; C096363:proteinase, foamy virus