An in vitro method for selective detection of free monomeric ubiquitin by using a C-terminally biotinylated form of ubiquitin.
In an effort to design a selective assay allowing detection of free monomeric ubiquitin, an approach based on a C-terminally biotinylated form of ubiquitin is proposed. In the form of a polyubiquitin chain, ubiquitin marks proteins for degradation by the 26S proteasome. This covalently attached signal is assembled from multiple ubiquitins linked to each other via the C-terminus of one ubiquitin and the epsilon-amine of Lys48 of another ubiquitin. In the present study, a form of ubiquitin having the C-terminus modified with the addition of a biotinylation peptide tag was prepared. After expression, this modified ubiquitin was biotinylated in vitro using recombinant biotin ligase. Biotinylated ubiquitin was further purified using affinity chromatography on immobilized monovalent avidin. This tagged form of ubiquitin is blocked at the C-terminus and therefore can only act as an acceptor (Lys-48 donor) in polyubiquitin chain synthesis. In vitro enzymatic assembly of multiubiquitin chains from biotinylated monoubiquitin and natural monoubiquitin is demonstrated by Western blot analysis using horseradish peroxidase-conjugated streptavidin. Data obtained with this assay indicate potential uses of the C-terminally biotinylated form of ubiquitin for selective detection of monoubiquitin contamination in a cell extract experimentally depleted of ubiquitin, i.e. lysate Fraction II. Cell-free systems established for in vitro examination of ubiquitin involvement in proteolytic processes usually employ Fraction II, which should be essentially ubiquitin-free. It is suggested that the assay using biotinylated monoubiquitin can be useful to exclude the possibility that ubiquitin contamination of laboratory prepared lysate Fraction II accounts for protein degradation in this fraction.
Staszczak
Magdalena
M
Department of Biochemistry, Maria Curie-Skłodowska University, pl. M. Curie-Skłodowskiej 3, 20-031 Lublin, Poland. magda@hermes.umcs.lublin.pl
D000818:Animals; D001360:Avidin / Q000378:metabolism; D019921:Biotinylation; D015153:Blotting, Western; D002474:Cell-Free System / Q000737:chemistry; D002846:Chromatography, Affinity; D010455:Peptides / Q000378:metabolism; D025821:Polyubiquitin / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D025801:Ubiquitin / Q000032:analysis*; D014452:Ubiquitins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D010455:Peptides; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D014452:Ubiquitins; D025821:Polyubiquitin; D001360:Avidin