title: An Escherichia coli expression system for glutamyl endopeptidases optimized by complete suppression of autodegradation.
abstract: V8 protease (GluV8), a member of the glutamyl endopeptidase I family isolated from the V8 strain of Staphylococcus aureus, is widely used for proteome analysis because of its unique substrate specificity and resistance to detergents. We recently developed an Escherichia coli expression system for the production of GluV8 based on a technique that suppresses the autoproteolysis--the use of the prosequence of its homologue (GluSE) from Staphylococcus epidermidis as a chimeric form or the introduction of four substitutions in the prosequence of GluV8. In the current study, we refined this technique through five amino acid substitutions within the prosequence of GluV8 for complete suppression of the autodegradation. As a result, the recovery of GluV8 proform was enhanced to 20 fg/cell, which was comparable to the level of a constitutive inactive form of GluV8, indicating complete suppression of the autoproteolysis. This mutated propeptide was also effective for the expression of the mature sequence of the glutamyl endopeptidase from Staphylococcus warneri. The recombinant proteins were successfully converted to their active forms through a common cleavage mechanism mediated by thermolysin in vitro. This strategy may shed light on the way for the expression of the proteases that have been scarcely produced in E. coli to date.
authors.affiliation: Department of Oral Molecular Biology, Course of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8588, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Ono
authors.forename: Takayuki K
authors.initials: TK
authors.lastname: Nemoto
authors.forename: Yu
authors.initials: Y
authors.lastname: Shimoyama
authors.forename: Shigenobu
authors.initials: S
authors.lastname: Kimura
authors.forename: Yuko
authors.initials: Y
authors.lastname: Ohara-Nemoto
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000187:drug effects; D001671:Biochemistry / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology; D013212:Staphylococcus epidermidis / Q000201:enzymology; D013379:Substrate Specificity / Q000187:drug effects; D013820:Thermolysin / Q000494:pharmacology
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D013820:Thermolysin