Analysis of recombinant acylated pneumococcal surface adhesin A of Streptococcus pneumoniae by mass spectrometry

Barun K De; Adrian R Woolfitt; John R Barr; Maryam I Daneshvar; Jacquelyn S Sampson; Edwin W Ades; George M Carlone

doi:10.1016/j.abb.2003.07.001

Analysis of recombinant acylated pneumococcal surface adhesin A of Streptococcus pneumoniae by mass spectrometry

Arch Biochem Biophys. 2003 Nov 15;419(2):147-57. doi: 10.1016/j.abb.2003.07.001.

Authors

Barun K De¹, Adrian R Woolfitt, John R Barr, Maryam I Daneshvar, Jacquelyn S Sampson, Edwin W Ades, George M Carlone

Affiliation

¹ Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA.

PMID: 14592458
DOI: 10.1016/j.abb.2003.07.001

Abstract

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.

Publication types

Comparative Study
Evaluation Study
Validation Study

MeSH terms

Adhesins, Bacterial
Amino Acid Sequence
Bacterial Proteins / biosynthesis
Bacterial Proteins / chemistry
Bacterial Proteins / genetics
Carrier Proteins / biosynthesis*
Carrier Proteins / chemistry*
Carrier Proteins / classification
Carrier Proteins / genetics
Computer Simulation
Escherichia coli / chemistry
Escherichia coli / genetics
Escherichia coli / metabolism
Gene Expression Regulation, Bacterial
Lipoproteins / biosynthesis*
Lipoproteins / chemistry*
Lipoproteins / classification
Lipoproteins / genetics
Mass Spectrometry / methods
Membrane Transport Proteins*
Models, Molecular*
Molecular Sequence Data
Protein Conformation
Protein Denaturation
Recombinant Proteins / biosynthesis
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Spectrometry, Mass, Electrospray Ionization / methods*
Streptococcus pneumoniae / chemistry*
Streptococcus pneumoniae / genetics
Streptococcus pneumoniae / metabolism*
Trypsin / chemistry*

Substances

Adhesins, Bacterial
Bacterial Proteins
Carrier Proteins
Lipoproteins
Membrane Transport Proteins
PsaA protein, Streptococcus
Recombinant Proteins
Trypsin