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 approximately 41,000 and the fully active enzyme Mr approximately 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.
Boyle
M D
MD
Department of Microbiology and Immunology, Medical College of Ohio, 3055 Arlington Avenue, Toledo, OH 43614-5806, USA. Mboyle@mco.edu
Romer
T G
TG
Meeker
A K
AK
Sledjeski
D D
DD
D001426:Bacterial Proteins / Q000032:analysis* / Q000096:biosynthesis; D003470:Culture Media; D003546:Cysteine Endopeptidases / Q000032:analysis* / Q000096:biosynthesis; D004792:Enzyme Precursors / Q000032:analysis* / Q000096:biosynthesis; D007700:Kinetics; D013058:Mass Spectrometry; D013297:Streptococcus pyogenes / Q000201:enzymology* / Q000254:growth & development
D001426:Bacterial Proteins; D003470:Culture Media; D004792:Enzyme Precursors; D003546:Cysteine Endopeptidases; C042477:streptopain