title: Purification and characterization of Streptococcus pneumoniae palmitoylated pneumococcal surface adhesin A expressed in Escherichia coli.
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 ( approximately 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 ( approximately 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 [(3)H] 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.
authors.affiliation: Division of Bacterial and Mycotic Diseases, and Biotechnology Core Facility Branch, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA. bkd1@cdc.gov
authors.forename: B K
authors.initials: BK
authors.lastname: De
authors.forename: J S
authors.initials: JS
authors.lastname: Sampson
authors.forename: E W
authors.initials: EW
authors.lastname: Ades
authors.forename: R C
authors.initials: RC
authors.lastname: Huebner
authors.forename: D L
authors.initials: DL
authors.lastname: Jue
authors.forename: S E
authors.initials: SE
authors.lastname: Johnson
authors.forename: M
authors.initials: M
authors.lastname: Espina
authors.forename: A R
authors.initials: AR
authors.lastname: Stinson
authors.forename: D E
authors.initials: DE
authors.lastname: Briles
authors.forename: G M
authors.initials: GM
authors.lastname: Carlone
mesh_terms: D018829:Adhesins, Bacterial; D000276:Adjuvants, Immunologic / Q000008:administration & dosage; D000281:Administration, Intranasal; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001426:Bacterial Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology; D002352:Carrier Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology; D002772:Cholera Toxin / Q000008:administration & dosage / Q000276:immunology; D003902:Detergents / Q000737:chemistry; D004306:Dose-Response Relationship, Immunologic; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005260:Female; D007074:Immunoglobulin G / Q000097:blood; D008074:Lipoproteins / Q000008:administration & dosage / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology; D026901:Membrane Transport Proteins; D051379:Mice; D008808:Mice, Inbred CBA; D008970:Molecular Weight; D010169:Palmitic Acids / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics; D012463:Saliva / Q000737:chemistry; D013296:Streptococcus pneumoniae / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000276:immunology
chemical_list: D018829:Adhesins, Bacterial; D000276:Adjuvants, Immunologic; D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003902:Detergents; D007074:Immunoglobulin G; D008074:Lipoproteins; D026901:Membrane Transport Proteins; D010169:Palmitic Acids; D021382:Protein Sorting Signals; C085000:PsaA protein, Streptococcus; D014614:Vaccines, Synthetic; D002772:Cholera Toxin