title: Characterization of the glutamyl endopeptidase from Staphylococcus aureus expressed in Escherichia coli.
abstract: V8 protease, a member of the glutamyl endopeptidase I family, of Staphylococcus aureus V8 strain (GluV8) is widely used for proteome analysis because of its unique substrate specificity and resistance to detergents. In this study, an Escherichia coli expression system for GluV8, as well as its homologue from Staphylococcus epidermidis (GluSE), was developed, and the roles of the prosegments and two specific amino acid residues, Val69 and Ser237, were investigated. C-terminal His(6)-tagged proGluSE was successfully expressed from the full-length sequence as a soluble form. By contrast, GluV8 was poorly expressed by the system as a result of autodegradation; however, it was efficiently obtained by swapping its preprosegment with that of GluSE, or by the substitution of four residues in the GluV8 prosequence with those of GluSE. The purified proGluV8 was converted to the mature form in vitro by thermolysin treatment. The prosegment was essential for the suppression of proteolytic activity, as well as for the correct folding of GluV8, indicating its role as an intramolecular chaperone. Furthermore, the four amino acid residues at the C-terminus of the prosegment were sufficient for both of these roles. In vitro mutagenesis revealed that Ser237 was essential for proteolytic activity, and that Val69 was indispensable for the precise cleavage by thermolysin and was involved in the proteolytic reaction itself. This is the first study to express quantitatively GluV8 in E. coli, and to demonstrate explicitly the intramolecular chaperone activity of the prosegment of glutamyl endopeptidase I.
authors.affiliation: Department of Oral Molecular Biology, Course of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, Japan. tnemoto@nagasaki-u.ac.jp
authors.forename: Takayuki K
authors.initials: TK
authors.lastname: Nemoto
authors.forename: Yuko
authors.initials: Y
authors.lastname: Ohara-Nemoto
authors.forename: Toshio
authors.initials: T
authors.lastname: Ono
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kobayakawa
authors.forename: Yu
authors.initials: Y
authors.lastname: Shimoyama
authors.forename: Shigenobu
authors.initials: S
authors.lastname: Kimura
authors.forename: Takashi
authors.initials: T
authors.lastname: Takagi
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics; D013212:Staphylococcus epidermidis / Q000201:enzymology / Q000235:genetics; D013329:Structure-Activity Relationship; D013820:Thermolysin / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012694:Serine; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D013820:Thermolysin