Using deubiquitylating enzymes as research tools.
Ubiquitin is synthesized in eukaryotes as a linear fusion with a normal peptide bond either to itself or to one of two ribosomal proteins and, in the latter case, enhances the yield of these ribosomal proteins and/or their incorporation into the ribosome. Such fusions are cleaved rapidly by a variety of deubiquitylating enzymes. Expression of heterologous proteins as linear ubiquitin fusions has been found to significantly increase the yield of unstable or poorly expressed proteins in either bacterial or eukaryotic hosts. If expressed in bacterial cells, the fusion is not cleaved due to the absence of deubiquitylating activity and can be purified intact. We have developed an efficient expression system, utilizing the ubiquitin fusion technique and a robust deubiquitylating enzyme, which allows convenient high yield and easy purification of authentic proteins. An affinity purification tag on both the ubiquitin fusion and the deubiquitylating enzyme allows their easy purification and the easy removal of unwanted components after cleavage, leaving the desired protein as the only soluble product. Ubiquitin is also conjugated to epsilon amino groups in lysine side chains of target proteins to form a so-called isopeptide linkage. Either a single ubiquitin can be conjugated or other lysines within ubiquitin can be acceptors for further conjugation, leading to formation of a branched, isopeptide-linked ubiquitin chain. Removal of these ubiquitin moieties or chains in vitro would be a valuable tool in the ubiquitinologists tool kit to simplify downstream studies on ubiquitylated targets. The robust deubiquitylating enzyme described earlier is also very useful for this task.
Baker
Rohan T
RT
Molecular Genetics Group, Division of Molecular Medicine, John Curtin School of Medical Research, Australian National University, Canberra ACT 0200, Australia.
Catanzariti
Ann-Maree
AM
Karunasekara
Yamuna
Y
Soboleva
Tatiana A
TA
Sharwood
Robert
R
Whitney
Spencer
S
Board
Philip G
PG
D000595:Amino Acid Sequence; D000818:Animals; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012965:Sodium Chloride; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D064570:Ubiquitin-Specific Proteases
D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D012965:Sodium Chloride; D010450:Endopeptidases; D064570:Ubiquitin-Specific Proteases; C091939:ubiquitin isopeptidase