title: A novel strategy to over-express and purify homologous proteins from Streptococcus pneumoniae.
abstract: Functional studies of Streptococcus pneumoniae virulence factors are facilitated by the development of complementation/mutagenesis systems. These methods usually result in poor expression yields; therefore, biochemical and structural/functional characterizations are mostly performed with proteins expressed and purified from heterologous systems (e.g. Escherichia coli). However, heterologous expression does not guarantee correct protein structure and function. In this work, we developed a method to over-express and purify homologous proteins from S. pneumoniae. The system relies on the combined use of the shuttle plasmid pMU1328 and a natural constitutive pneumococcal promoter, P(96). Efficient over-expression of secreted, membrane or surface anchored proteins, either wild type or mutant, was achieved. As proof of principle the S. pneumoniae pilus-1 backbone RrgB was successfully purified as a His-tag secreted protein (RrgB-His_SP) from pneumococcal culture supernatants. N-terminal sequencing and mass spectrometry analysis of RrgB-His_SP allowed the determination of the leader sequence cleavage site in pneumococcus, while proteolysis studies confirmed the stability of RrgB-His_SP to trypsin digestion. The data presented here support the use of this novel homologous expression method for all S. pneumoniae proteins for which extensive characterization studies are planned. Moreover, given the promiscuity of the pMU1328 replicon, this system could be used in diverse bacterial species.
authors.affiliation: Novartis Vaccines and Diagnostics Research Center, 53100 Siena, Italy.
authors.forename: Morena
authors.initials: M
authors.lastname: Lo Sapio
authors.forename: Markus
authors.initials: M
authors.lastname: Hilleringmann
authors.forename: Michèle A
authors.initials: MA
authors.lastname: Barocchi
authors.forename: Monica
authors.initials: M
authors.lastname: Moschioni
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012093:Replicon / Q000235:genetics; D017384:Sequence Deletion; D013296:Streptococcus pneumoniae / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins