Characterization of a Helicobacter pylori vaccine candidate by proteome techniques.
In a previous two-dimensional (2D) gel electrophoretic study of protein antigens of the gastric pathogen, Helicobacter pylori recognized by human sera, one of the highly and consistently reactive antigens, a protein with Mr of approximately 30,000 (Spot 15) seemed to be of special interest because of low yields on N-terminal protein sequencing. This suggested possible N-terminal modification, as the N-terminal sequence analysis of this 30,000 protein (Spot 15) did not provide a definitive match within the H. pylori genomic database. This protein was isolated by 2D polyacrylamide gel electrophoresis, evaluated by liquid chromatography-mass spectrometry, and found to consist of two related species of approximately 28,100 and 26,500. In parallel, the proteins within this spot were digested in situ with the endoprotease Lys-C. Analysis of the Lys-C digest by matrix-assisted laser desorption time-of-flight mass spectrometry, peptide mapping, and sequence analysis was conducted. Comparison of the mass and sequence of the Lys-C peptides with those derived from a H. pylori genomic library identified an open reading frame of approximately 300 base pairs as the source of the Spot 15 protein. This corresponded to HP0175 in the recently reported H. pylori genome sequence, an open reading frame with some homology to Campylobacter jejeuni cell binding protein 2. Mass spectral and sequence analysis indicated that Spot 15 was a processed product generated by proteolytic cleavage at both the carboxy and amino termini of the 34 open reading frame precursor.
McAtee
C P
CP
Genelabs Technologies, Redwood City, CA 94063, USA.
Lim
M Y
MY
Fung
K
K
Velligan
M
M
Fry
K
K
Chow
T P
TP
Berg
D E
DE
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry*; D001428:Bacterial Vaccines / Q000737:chemistry*; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D016480:Helicobacter pylori / Q000276:immunology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000942:Antigens, Bacterial; D001428:Bacterial Vaccines; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase