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

Academic Article

Abstract

  • All Streptococcus pneumoniae isolates tested to date express a species- common lipoprotein designated as pneumococcal surface adhesin A (PsaA). This protein is cell-associated, hydrophobic, immunogenic, and genetically conserved. It is currently under investigation as a potential component in third-generation 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 (~10 mg/l of fermentation culture) using Borrelia burgdorferi outer surface protein A (OspA, a hydrophobic lipoprotein) signal peptide. By Western blot analysis, the chimeric rPsaA (~34 kDa) was detected in the cell lysate using anti-PsaA antibodies. It was partially purified by extracting the cell pellet with PBS/Triton X(R)-114 buffers, followed by anion exchange filter chromatography. A trypsin digestion profile of rPsaA closely resembled that of the native protein, as revealed by SDS-PAGE/silver staining. Lipidation of rPsaA was confirmed by labeling recombinant E. coli cells with [3H] palmitic acid and analyzing the labeled E. coli cells by Western blotting coupled with autoradiography. Further, analysis of purified rPsaA by mass spectrometry (MALDI-TOF) revealed a heterogenous spectrum with a major peak (M+H)+1 of mass 33,384 Da (theoretical mass of palmitoylated rPsaA = 33,361 Da). Purified rPsaA was immunogenic in CBA/NCAHN-XID female mice following intranasal immunization with or without adjuvant, as determined by measurement of anti-PsaA serum IgG levels. These anti-PsaA antibodies reacted with both native and rPsaA polypeptides. Our data strongly suggest that E. coli-expressed rPsaA is palmitoylated and closely resembles the native protein in structure and immunogenicity. It was also observed to elicit measurable protection against nasopharyngeal carriage with S. pneumoniae. (C) 2000 Elsevier Science Ltd.
  • Authors

    Published In

  • Vaccine  Journal
  • Digital Object Identifier (doi)

    Pubmed Id

  • 6127709
  • Author List

  • De BK; Sampson JS; Ades EW; Huebner RC; Jue DL; Johnson SE; Espina M; Stinson AR; Briles DE; Carlone GM
  • Start Page

  • 1811
  • End Page

  • 1821
  • Volume

  • 18
  • Issue

  • 17