Strategies for assaying deubiquitinating enzymes.
A general method for assaying deubiquitinating enzymes (DUBs) has been developed. This new method employs an indirect enzyme assay for determining the activity of DUBs using a linear fusion of polyHis-glutathione-S-transferase-ubiquitin-ecotin (His-GST-Ub-ecotin) as a substrate. Because ecotin, a trypsin inhibitor protein from Escherichia coli, is heat stable, the activity of DUBs can be assayed indirectly by determining the ability of ecotin to inhibit trypsin after incubation of any DUB with His-GST-Ub-ecotin followed by heating at 100 degrees. In the substrate construction, His-GST fusion to Ub was used for facilitation of the substrate purification as well as for assisting the heat precipitation of His-GST-Ub and uncleaved His-GST-Ub-ecotin, as Ub itself is also heat stable. This method can also be used for assaying the proteases that process Ub-like proteins (Ubls) using the substrates, in which Ub is replaced by Ubls.
Kang
Sung Hwan
SH
School of Biological Sciences, Seoul National University, Seoul 151-742, Korea.
Park
Jung Jun
JJ
Chung
Sung Soo
SS
Bang
Ok Sun
OS
Chung
Chin Ha
CH
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D010450:Endopeptidases / Q000032:analysis* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D033903:Periplasmic Proteins / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin; D014361:Trypsin Inhibitors; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism
C039076:Eco protein, E coli; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D011993:Recombinant Fusion Proteins; D014361:Trypsin Inhibitors; D025801:Ubiquitin; C033223:polyhistidine; D006639:Histidine; D010450:Endopeptidases; D014357:Trypsin; C091939:ubiquitin isopeptidase; D005978:Glutathione