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Characterization of the Glutamyl Endopeptidase From Staphylococcus Aureus Expressed in Escherichia Coli

Characterization of the Glutamyl Endopeptidase From Staphylococcus Aureus Expressed in Escherichia Coli

Takayuki K Nemoto, Yuko Ohara-Nemoto, Toshio Ono, Takeshi Kobayakawa, Yu Shimoyama, Shigenobu Kimura, Takashi Takagi

FEBS J. 2008 Feb;275(3):573-87.

PMID: 18199287

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP66676435-A	Endoproteinase Glu-C from Staphylococcus aureus V8		66676-43-5	Price

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