Production and characterization of the functional fragment of pneumococcalsurface protein A

Pneumococcal surface protein A (PspA) is present on the cell wall of Strept ococcus pneumoniae pathogen and has an antigenetically variable N-terminal domain. This aminoterminal domain is essential for full pneumococcal virule nce, and monoclonal antibodies raised against it protect mice against pneum ococcal infections, We have cloned and expressed a 34-kDa N-terminal fragme nt of PspA in Escherichia coli in a soluble form using the T7 RNA polymeras e pET-20b vector system. Nickel chelate affinity purification followed by s ize exclusion and anion exchange chromatography yielded large amounts of pu re and homogeneous protein. Analytical ultracentrifugation sedimentation ve locity band and boundary studies showed that the molecule was present in aq ueous solutions in a monomeric form with an axial shape ratio of approximat ely 1:12, typical of fibrous proteins. Sequence analyses indicated an alpha -helical coiled-coil structure for this monomeric molecule with only few lo op-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 par t of the PspA molecule are proposed. The evident highly charged and polar c haracter of the surface of the modeled structures suggests functional prope rties of PspA that are related to the prevention of S. pneumoniae interacti ons with the host complement system. (C) 2000 Academic Press.