Production and characterization of the functional fragment of pneumococcal surface protein A.
Pneumococcal surface protein A (PspA) is present on the cell wall of Streptococcus pneumoniae pathogen and has an antigenetically variable N-terminal domain. This aminoterminal domain is essential for full pneumococcal virulence, and monoclonal antibodies raised against it protect mice against pneumococcal infections. We have cloned and expressed a 34-kDa N-terminal fragment of PspA in Escherichia coli in a soluble form using the T7 RNA polymerase pET-20b vector system. Nickel chelate affinity purification followed by size exclusion and anion exchange chromatography yielded large amounts of pure and homogeneous protein. Analytical ultracentrifugation sedimentation velocity band and boundary studies showed that the molecule was present in aqueous solutions in a monomeric form with an axial shape ratio of approximately 1:12, typical of fibrous proteins. Sequence analyses indicated an alpha-helical coiled-coil structure for this monomeric molecule with only few loop-type breaks in helicity. The mostly alpha-helical structure of this PspA construct was consistent with circular dichroism spectroscopy data. Based on the ultracentrifugation studies, the circular dichroism spectra, and the PspA's sequence analyses, two structural models for the amino-terminal part of the PspA molecule are proposed. The evident highly charged and polar character of the surface of the modeled structures suggests functional properties of PspA that are related to the prevention of S. pneumoniae interactions with the host complement system.
Jedrzejas
M J
MJ
Department of Microbiology, University of Alabama at Birmingham, 933 19th Street South, 545 CHSB-19, Birmingham, Alabama, 35294, USA. jedrzejas@uab.edu
Hollingshead
S K
SK
Lebowitz
J
J
Chantalat
L
L
Briles
D E
DE
Lamani
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D000954:Antigens, Surface / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D014774:Virulence
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001426:Bacterial Proteins; D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; C051592:pneumococcal surface protein A