A functional proteomic analysis of secreted fibrinolytic enzymes from Bacillus subtilis 168 using a combined method of two-dimensional gel electrophoresis and zymography.
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.
Park
Sung Goo
SG
Proteome Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, Yusung, Taejon, South Korea.
Kho
Chang Won
CW
Cho
Sayeon
S
Lee
Do Hee
DH
Kim
Seung Ho
SH
Park
Byoung Chul
BC
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
D001426:Bacterial Proteins; D020543:Proteome; D010450:Endopeptidases; D012697:Serine Endopeptidases; C104015:WprA protein, Bacillus subtilis