tailieunhanh - Báo cáo khoa học: Characterization of the glutamyl endopeptidase from Staphylococcus aureus expressed in Escherichia coli

V8 protease, a member of the glutamyl endopeptidase I family, ofStaphy-lococcus 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 coliexpression system for GluV8, as well as its homologue fromStaphylococcus epidermidis(GluSE), was developed, and the roles of the prosegments and two specific amino acid residues, Val69 and Ser237, were investigated. | ỊFEBS Journal Characterization of the glutamyl endopeptidase from Staphylococcus aureus expressed in Escherichia coli Takayuki K. Nemoto1 Yuko Ohara-Nemoto1 Toshio Ono1 Takeshi Kobayakawa1 Yu Shimoyama2 Shigenobu Kimura2 and Takashi Takagi3 1 Department of OralMolecular Biology Course of Medicaland DentalSciences Nagasaki University Graduate Schoolof Biomedical Sciences Japan 2 Department of OralMicrobiology Iwate MedicalUniversity Schoolof Dentistry Morioka Japan 3 Department of DevelopmentalBiology and Neurosciences Graduate Schoolof Life Sciences Tohoku University Sendai Japan Keywords chaperone glutamyl endopeptidase Staphylococcus aureus Staphylococcus epidermidis V8 protease Correspondence T. K. Nemoto Department of Oral Molecular Biology Course of Medicaland Dental Sciences Nagasaki University 1-7-1 Sakamoto Nagasaki 852-8588 Japan Fax 81 95 819 7642 Tel 81 95 819 7640 E-mail tnemoto@ Received 2 November 2007 revised 6 December 2007 accepted 7 December 2007 doi 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 His6-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 .

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