Purification and characterization of Streptococcus pneumoniae palmitoylated pneumococcal surface adhesin A expressed in Escherichia coli

All Streptococcus pneumoniae isolates tested to date express a species-comm on lipoprotein designated as pneumococcal surface adhesin A (PsaA). This pr otein is cell-associated, hydrophobic, immunogenic, and genetically conserv ed. It is currently under investigation as a potential component in third-g eneration pneumococcal vaccine formulations. To overcome the problem of low -level expression of native hydrophobic PsaA in S, pneumoniae, and also of the recombinant PsaA (rPsaA) in Escherichia coli, we generated a stable E. coli construct expressing functional palmitoylated rPsaA (similar to 10 mg/ l of fermentation culture) using Borrelia burgdorferi outer surface protein A (OspA, a hydrophobic lipoprotein) signal peptide. By Western blot analys is, the chimeric rPsaA (similar to 34 kDa) was detected in the cell lysate using anti-PsaA antibodies. It was partially purified by extracting the cel l pellet with PBS/Triton X-R-114 buffers, followed by anion exchange filter chromatography. A trypsin digestion profile of rPsaA closely resembled tha t of the native protein, as revealed by SDS-PAGE/silver staining. Lipidatio n of rPsaA was confirmed by labeling recombinant E. coli cells with [H-3] p almitic acid and analyzing the labeled E. coil cells by Western blotting co upled with autoradiography. Further, analysis of purified rPsaA by mass spe ctrometry (MALDI-TOF) revealed a heterogenous spectrum with a major peak (M + H)(+1) of mass 33,384 Da (theoretical mass of palmitoylated rPsaA = 33,3 61 Da). Purified rPsaA was immunogenic in CBA/NCAHN-XID female mice followi ng intranasal immunization with or without adjuvant, as determined by measu rement of anti-PsaA serum IgG levels, These anti-PsaA antibodies reacted wi th both native and rPsaA polypeptides. Our data strongly suggest that E. co li-expressed rPsaA is palmitoylated and closely resembles the native protei n in structure and immunogenicity. It was also observed to elicit measurabl e protection against nasopharyngeal carriage with S. pneumoniae. Published by Elsevier Science Ltd.