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.
De
Barun K
BK
Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA.
Woolfitt
Adrian R
AR
Barr
John R
JR
Daneshvar
Maryam I
MI
Sampson
Jacquelyn S
JS
Ades
Edwin W
EW
Carlone
George M
GM
D018829:Adhesins, Bacterial; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000145:classification / Q000235:genetics; D003198:Computer Simulation; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008074:Lipoproteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000145:classification / Q000235:genetics; D013058:Mass Spectrometry / Q000379:methods; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013296:Streptococcus pneumoniae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry*
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D002352:Carrier Proteins; D008074:Lipoproteins; D026901:Membrane Transport Proteins; C085000:PsaA protein, Streptococcus; D011994:Recombinant Proteins; D014357:Trypsin