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Analysis of recombinant acylated pneumococcal surface adhesin A of Streptococcus pneumoniae by mass spectrometry.
De, Barun K; Woolfitt, Adrian R; Barr, John R; Daneshvar, Maryam I; Sampson, Jacquelyn S; Ades, Edwin W; Carlone, George M.
Afiliación
  • De BK; Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA.
Arch Biochem Biophys ; 419(2): 147-57, 2003 Nov 15.
Article en En | MEDLINE | ID: mdl-14592458
Streptococcus pneumoniae pneumococcal surface adhesin A (PsaA) is a species-common, immunogenic surface lipoprotein. In this study, the psaA gene was expressed as a nonfusion acylated protein in an Escherichia coli expression system. Yields of pure recombinant PsaA (rPsaA) were 8-10 mg/liter of fermentation culture. Analysis of rPsaA tryptic digests by HPLC-electrospray mass spectrometry (MS) confirmed 98% of the expected protein sequence. GC/MS data demonstrated very similar acylation of native and rPsaA by C12:0-C22:0 fatty acids, with C16 and C18 predominating. Negative ion electrospray MS/MS analysis of the rPsaA lipid anchor released by Pronase-E confirmed that the structure was based on an N-terminal palmitoylcysteine (Pam(3)Cys). Electrospray MS heterogeneity analysis of intact rPsaA indicated that all of the observed heterogeneity could be accounted for by the fatty acid distributions. The availability of well-characterized rPsaA will facilitate the continued research and development of protein-based vaccines for the prevention of pneumococcal disease.
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Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas de Transporte de Membrana / Streptococcus pneumoniae / Tripsina / Proteínas Portadoras / Modelos Moleculares / Espectrometría de Masa por Ionización de Electrospray / Lipoproteínas Tipo de estudio: Evaluation_studies Idioma: En Revista: Arch Biochem Biophys Año: 2003 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos
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Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas de Transporte de Membrana / Streptococcus pneumoniae / Tripsina / Proteínas Portadoras / Modelos Moleculares / Espectrometría de Masa por Ionización de Electrospray / Lipoproteínas Tipo de estudio: Evaluation_studies Idioma: En Revista: Arch Biochem Biophys Año: 2003 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos