title: Functional expression, purification, and biochemical properties of subtilase SprP from Pseudomonas aeruginosa.
abstract: The Pseudomonas aeruginosa genome encodes a variety of different proteolytic enzymes several of which play an important role as virulence factors. Interestingly, only two of these proteases are predicted to belong to the subtilase family and we have recently studied the physiological role of the subtilase SprP. Here, we describe the functional overexpression of SprP in Escherichia coli using a novel expression and secretion system. We show that SprP is autocatalytically activated by proteolysis and exhibits optimal activity at 50°C in a pH range of 7-8. We also demonstrate a significant increase in sprP promoter activity upon growth of P. aeruginosa at 43°C indicating a role for SprP in heat shock response.
authors.affiliation: Institute of Molecular Enzyme Technology, Heinrich-Heine-University Duesseldorf, Forschungszentrum Juelich, D-52426, Juelich, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Pelzer
authors.affiliation: Institute of Biochemistry, Heinrich-Heine-University Duesseldorf, Universitaetsstr. 1, D-40225, Duesseldorf, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Schwarz
authors.affiliation: Institute of Molecular Enzyme Technology, Heinrich-Heine-University Duesseldorf, Forschungszentrum Juelich, D-52426, Juelich, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Knapp
authors.affiliation: Institute of Molecular Enzyme Technology, Heinrich-Heine-University Duesseldorf, Forschungszentrum Juelich, D-52426, Juelich, Germany.
authors.forename: Astrid
authors.initials: A
authors.lastname: Wirtz
authors.affiliation: Institute of Molecular Enzyme Technology, Heinrich-Heine-University Duesseldorf, Forschungszentrum Juelich, D-52426, Juelich, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Wilhelm
authors.affiliation: Institute of Biochemistry, Heinrich-Heine-University Duesseldorf, Universitaetsstr. 1, D-40225, Duesseldorf, Germany.
authors.forename: Sander
authors.initials: S
authors.lastname: Smits
authors.affiliation: Institute of Biochemistry, Heinrich-Heine-University Duesseldorf, Universitaetsstr. 1, D-40225, Duesseldorf, Germany.
authors.forename: Lutz
authors.initials: L
authors.lastname: Schmitt
authors.affiliation: Institute of Molecular Enzyme Technology, Heinrich-Heine-University Duesseldorf, Forschungszentrum Juelich, D-52426, Juelich, Germany.
authors.forename: Horst
authors.initials: H
authors.lastname: Funken
authors.affiliation: Institute of Molecular Enzyme Technology, Heinrich-Heine-University Duesseldorf, Forschungszentrum Juelich, D-52426, Juelich, Germany.|Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Juelich GmbH, D-52426, Juelich, Germany.
authors.forename: Karl-Erich
authors.initials: KE
authors.lastname: Jaeger
mesh_terms: D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling; D006863:Hydrogen-Ion Concentration; D059748:Proteolysis; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D013381:Subtilisins
keywords: Autocatalytic processing; Pseudomonas aeruginosa; heat induction; subtilase SprP.