title: Optimizing expression of Streptococcus pneumoniae surface protein a, PspA: serocross-reactivity within families of antisera induced against clades 1 and 3.
abstract: Streptococcus pneumoniae is the agent responsible for infections such as pneumonia, otitis media, and meningitis. Among virulence factors, the Pneumococcal surface protein A (PspA) has been shown to be immunogenic and protective in mice, and is thus a good vaccine candidate. PspA has been classified into 6 clades and 3 families. Initially, pspA fragments, clades 1 and 3, were cloned into the pAE-6His expression vector. Proteins were expressed in Escherichia coli BL21(DE3) and purified by affinity and anion exchange chromatographies, with a yield of 11 mg/l of culture. Due to plasmid instability in E. coli, another construct using pspA1 was obtained based on pET-37b(+), which was shown to be stable in E. coli and increased the yield approximately 3-fold. Our results show good conditions for scale-up. Sera from immunized mice recognized PspA in total extracts of S. pneumoniae strains: anti-rPspA1p sera recognized native PspA clades 1 (+++), 2 (++) and 4 (+) and anti-rPspA3p sera recognized PspA clades 1 (+), 2 (+), 3 (+++) and 4 (+). The cross-reactivity pattern obtained confirms the notion that proteins from both families should be included for development of a broad-coverage vaccine; lower-cross reactivity between rPspAs of family 2 indicates that it may be necessary to include 2 proteins from this family.
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, Av. Vital Brasil 1500, Sao Paulo, SP, 05503-900, Brazil.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Silva
authors.forename: Joaquin
authors.initials: J
authors.lastname: Cabrera-Crespo
authors.forename: Maria E
authors.initials: ME
authors.lastname: Sbrogio-Almeida
authors.forename: Eliane N
authors.initials: EN
authors.lastname: Miyaji
authors.forename: Paulo L
authors.initials: PL
authors.lastname: Ho
authors.forename: Luciana C C
authors.initials: LC
authors.lastname: Leite
authors.forename: Alexandre P Y
authors.initials: AP
authors.lastname: Lopes
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood* / Q000276:immunology*; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D003429:Cross Reactions / Q000276:immunology; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D011994:Recombinant Proteins; C051592:pneumococcal surface protein A