Mapping and identification of Brucella melitensis proteins by two-dimensional electrophoresis and microsequencing.
Two-dimensional (2-D) gel electrophoresis was used to map Brucella melitensis proteins. The 2-D proteins map of B. melitensis B115 revealed 595 silver-stained protein spots separated by both isoelectric point and molecular mass. Twenty-five proteins were identified either by immunoblotting using monoclonal antibodies (MAbs) or by N-terminal microsequencing. The protein spots identified by MAbs were the 89 kDa outer membrane protein, DnaK, bacterioferritin, CP24, and BP26. Some spots were identified by N-terminal microsequencing as proteins whose sequences had been reported previously from Brucella, such as three heat-shock proteins, namely DnaK, GroEL and GroES; bacterioferritin; Cu-Zn superoxide dismutase; and the 50S ribosomal protein L7/L12. Other proteins had amino acid sequences homologous with those of various proteins from other bacteria found in protein databases: ClpP; the 10K-S protein; the ORFU phosphoprotein; succinyl-CoA synthetase alpha sub-unit; an inorganic pyrophosphatase; the Fe and/or Mn superoxide dismutase; the nucleoside diphosphate kinase, an amino acid ABC type transporter, and an electron transfer flavoprotein small subunit. Seven proteins were identified with N-terminal sequences not yet reported in databases. The 2-D map established in this study will be the basis for comparative studies of protein expression in Brucella.
Teixeira-Gomes
A P
AP
Laboratoire de Pathologie Infectieuse et Immunologie, Institut National de la Recherche Agronomique, Nouzilly, France.
Cloeckaert
A
A
Bézard
G
G
Dubray
G
G
Zygmunt
M S
MS
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000737:chemistry; D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry*; D017347:Brucella melitensis / Q000737:chemistry*; D018835:Chaperonin 10 / Q000032:analysis / Q000737:chemistry; D018834:Chaperonin 60 / Q000032:analysis / Q000737:chemistry; D003573:Cytochrome b Group / Q000032:analysis / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D029968:Escherichia coli Proteins; D005293:Ferritins / Q000032:analysis / Q000737:chemistry; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis / Q000737:chemistry; D015151:Immunoblotting; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D016415:Sequence Alignment; D017421:Sequence Analysis; D016622:Silver Staining
D000911:Antibodies, Monoclonal; C106040:BP26 protein, Brucella; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C102461:CP24 protein, Brucella melitensis; D018835:Chaperonin 10; D018834:Chaperonin 60; D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D005293:Ferritins; C018032:bacterioferritin; C063842:dnaK protein, E coli