title: A functional proteomic analysis of secreted fibrinolytic enzymes from Bacillus subtilis 168 using a combined method of two-dimensional gel electrophoresis and zymography.
abstract: Here we describe a proteomic approach to detect fibrinolytic enzymes from the culture supernatant of Bacillus subtilis 168. Following isoelectric focusing without dithiothreitol, two gels, one for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the other for zymography, were run in parallel. After silver staining of SDS-PAGE and activity staining of zymography gel, the two gels were superimposed to detect protein spots that coincided with clear zones on the zymography gel. We identified four protein spots and characterized them with matrix-assisted laser desorption/ionization mass spectrometry. Database search revealed that four spots contained at least one of the extracellular serine proteases such as WprA and Vpr. This combined method of two-dimensional gel and zymography can be used as a powerful tool to detect proteases from various organisms.
authors.affiliation: Proteome Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, Yusung, Taejon, South Korea.
authors.forename: Sung Goo
authors.initials: SG
authors.lastname: Park
authors.forename: Chang Won
authors.initials: CW
authors.lastname: Kho
authors.forename: Sayeon
authors.initials: S
authors.lastname: Cho
authors.forename: Do Hee
authors.initials: DH
authors.lastname: Lee
authors.forename: Seung Ho
authors.initials: SH
authors.lastname: Kim
authors.forename: Byoung Chul
authors.initials: BC
authors.lastname: Park
mesh_terms: D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins; D002417:Cattle; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification; D005342:Fibrinolysis; D066298:In Vitro Techniques; D008970:Molecular Weight; D020543:Proteome / Q000235:genetics / Q000302:isolation & purification*; D012697:Serine Endopeptidases / Q000235:genetics / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001426:Bacterial Proteins; D020543:Proteome; D010450:Endopeptidases; D012697:Serine Endopeptidases; C104015:WprA protein, Bacillus subtilis