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

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.