Rapid purification method for the 26S proteasome from the filamentous fungus Trichoderma reesei.
We have developed a fast and simple two column chromatographic method for the purification of the 26S proteasome from the filamentous fungus Trichoderma reesei that simplifies the overall procedure and reduces the purification time from 5 to 2.5 days. The combination of only the anionic exchange POROS HQ column (Applied Biosystems) together with a size exclusion column has not been used previously for proteasome purification. The purified complex was analysed further by two-dimensional electrophoresis (2DE) and examined by transmission electron microscopy (TEM). A total of 102 spots separated by 2DE were identified by mass spectrometry using cross-species identification (CSI) or an in-house custom-made protein database derived from the T. reesei sequencing project. Fifty-one spots out of 102 represented unique proteins. Among them, 30 were from the 20S particle and eight were from the 19S particle. In addition, seven proteasome-interacting proteins as well as several non-proteasome related proteins were identified. Co-purification of the 19S regulatory particle was confirmed by TEM and Western blotting. The rapidity of the purification procedure and largely intact nature of the complex suggest that similar procedure may be applicable to the isolation and purification of the other protein complexes.
Kautto
Liisa
L
Department of Chemistry and Biomolecular Sciences, Macquarie University, Herring Road, Sydney, NSW 2109, Australia. lkautto@bio.mq.edu.au
Grinyer
Jasmine
J
Birch
Debra
D
Kapur
Amit
A
Baker
Mark
M
Traini
Mathew
M
Bergquist
Peter
P
Nevalainen
Helena
H
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D015180:Electrophoresis, Gel, Two-Dimensional; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification*; D013058:Mass Spectrometry; D046529:Microscopy, Electron, Transmission; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000302:isolation & purification* / Q000648:ultrastructure; D040901:Proteomics / Q000379:methods*; D014242:Trichoderma / Q000201:enzymology*
D005656:Fungal Proteins; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease