title: Cloning, expression, purification, and characterization of Streptococcus pneumoniae IgA1 protease.
abstract: The IgA1 protease of Streptococcus pneumoniae is a Zn-metalloproteinase of 1964 amino acids that specifically cleaves the hinge region of IgA1, the predominant class of immunoglobulin present on mucosal membranes. This protease is associated to the bacterial cell surface via an N-terminal membrane anchor. Following proteolysis it is released in several forms of different molecular weight. Here, we describe the cloning, expression, and characterization of the enzymatic activity and immunogenicity of three fragments of IgA1 protease, including a large one lacking only the 103 N-terminal amino acids that constitute a typical prokaryotic signal sequence. Further, a proteolytically inactive mutant was generated by replacement of the glutamate residue with an alanine residue in the active site motif HExxH (1605-1609). This is the first report of recombinant active forms of S. pneumoniae IgA1 protease, which open the possibility of identifying specific inhibitors that could interfere with the mucosal colonization by pneumococcus. Moreover the inactive mutant could be considered as a candidate vaccine component.
authors.affiliation: Dipartimento di Scienze Biomediche, Università di Padova, Viale G. Colombo 3, 35121 Padova, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Romanello
authors.forename: M
authors.initials: M
authors.lastname: Marcacci
authors.forename: F
authors.initials: F
authors.lastname: Dal Molin
authors.forename: M
authors.initials: M
authors.lastname: Moschioni
authors.forename: S
authors.initials: S
authors.lastname: Censini
authors.forename: A
authors.initials: A
authors.lastname: Covacci
authors.forename: A G
authors.initials: AG
authors.lastname: Baritussio
authors.forename: C
authors.initials: C
authors.lastname: Montecucco
authors.forename: F
authors.initials: F
authors.lastname: Tonello
mesh_terms: D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D011018:Pneumonia, Pneumococcal / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000201:enzymology*
chemical_list: D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C021433:IgA-specific serine endopeptidase