Use of surface-enhanced laser desorption ionization protein chip system to analyze streptococcal exotoxin B activity secreted by Streptococcus pyogenes

Ciphergen surface-enhanced laser desorption ionization (SELDI) protein chip technology was used to analyze the secretion and autoactivation of the Streptococcus pyogenes cysteine protease SpeB. This method allowed rapid identification of both the zymogen form of the protein Mr∼41,000 and the fully a...

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Bibliographic Details
Published in:Journal of microbiological methods 2001-08, Vol.46 (2), p.87-97
Main Authors: Boyle, Michael D.P, Romer, Terence G, Meeker, Amanda K, Sledjeski, Darren D
Format: Article
Language:English
Subjects:
Bacterial diseases
Bacterial Proteins - analysis
Bacterial Proteins - biosynthesis
Bacteriology
Biological and medical sciences
Culture Media
Cysteine Endopeptidases - analysis
Cysteine Endopeptidases - biosynthesis
Enzyme Precursors - analysis
Enzyme Precursors - biosynthesis
exotoxin B
Experimental bacterial diseases and models
Fundamental and applied biological sciences. Psychology
Infectious diseases
Kinetics
Mass Spectrometry
Medical sciences
Microbiology
Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains
Protein chip system
Streptococcal exotoxin B
Streptococcus pyogenes
Streptococcus pyogenes - enzymology
Streptococcus pyogenes - growth & development
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Summary:Ciphergen surface-enhanced laser desorption ionization (SELDI) protein chip technology was used to analyze the secretion and autoactivation of the Streptococcus pyogenes cysteine protease SpeB. This method allowed rapid identification of both the zymogen form of the protein Mr∼41,000 and the fully active enzyme Mr∼28,500. SpeB production in culture supernatants was demonstrated to be growth-phase regulated and SpeB positive and negative variants of a blood passaged S. pyogenes isolate could readily be distinguished. In kinetic studies of the autoactivation of the zymogen form of SpeB, the sequential generation of four intermediates was detected before the accumulation of the fully active enzyme. The methods described enabled enhanced speed, use of lower sample volumes and concentrations, and a more complete molecular characterization of SpeB than allowed by existing methods of analysis using SDS-PAGE and Western immunoblotting.
ISSN:0167-7012
1872-8359
DOI:10.1016/S0167-7012(01)00279-2