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Using Deubiquitylating Enzymes as Research Tools

Baker, Rohan; Catanzariti, Ann-Maree; Karunasekara, Yamuna; Soboleva, Tatiana; Sharwood, Robert; Whitney, Spencer; Board, Philip

Description

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...[Show more]

dc.contributor.authorBaker, Rohan
dc.contributor.authorCatanzariti, Ann-Maree
dc.contributor.authorKarunasekara, Yamuna
dc.contributor.authorSoboleva, Tatiana
dc.contributor.authorSharwood, Robert
dc.contributor.authorWhitney, Spencer
dc.contributor.authorBoard, Philip
dc.date.accessioned2015-12-13T22:27:12Z
dc.date.available2015-12-13T22:27:12Z
dc.identifier.isbn0121828034
dc.identifier.urihttp://hdl.handle.net/1885/73843
dc.description.abstractUbiquitin 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 ε 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.
dc.publisherElsevier
dc.relation.ispartofUbiquitin and Protein Degradation
dc.relation.isversionof1st Edition
dc.subjectKeywords: amino acid; bacterial protein; hybrid protein; isopeptide; lysine; peptide; ribosome protein; unclassified drug; article; bacterial cell; cell fusion; chemical analysis; eukaryotic cell; gene expression system; heterologous expression; host cell; nucleoti
dc.titleUsing Deubiquitylating Enzymes as Research Tools
dc.typeBook chapter
local.description.notesImported from ARIES
local.description.refereedYes
dc.date.issued2005
local.identifier.absfor100104 - Genetically Modified Animals
local.identifier.ariespublicationMigratedxPub3855
local.type.statusPublished Version
local.contributor.affiliationBaker, Rohan, College of Medicine, Biology and Environment, ANU
local.contributor.affiliationCatanzariti, Ann-Maree, College of Medicine, Biology and Environment, ANU
local.contributor.affiliationKarunasekara, Yamuna, College of Medicine, Biology and Environment, ANU
local.contributor.affiliationSoboleva, Tatiana, College of Medicine, Biology and Environment, ANU
local.contributor.affiliationSharwood, Robert, College of Medicine, Biology and Environment, ANU
local.contributor.affiliationWhitney, Spencer, College of Medicine, Biology and Environment, ANU
local.contributor.affiliationBoard, Philip, College of Medicine, Biology and Environment, ANU
local.bibliographicCitation.startpage540
local.bibliographicCitation.lastpage554
local.identifier.doi10.1016/S0076-6879(05)98044-0
dc.date.updated2015-12-11T08:29:24Z
local.bibliographicCitation.placeofpublicationCalifornia
local.identifier.scopusID2-s2.0-27644595172
CollectionsANU Research Publications

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