title: The missing links to link ubiquitin: Methods for the enzymatic production of polyubiquitin chains.
abstract: Attachment of ubiquitin (Ub) as monoUb and polyUb chains of different lengths and linkages to proteins plays a dominant role in very different regulatory mechanisms. Therefore, the study of polyUb chains has assumed a central interest in biochemistry and structural biology. An essential step necessary to allow in vitro biochemical and structural studies of polyUbs is the production of their chains in high quantities and purity. This is not always an easy task and can be achieved both enzymatically and chemically. Previous reviews have covered chemical cross-linking exhaustively. In this review, we concentrate on the different approaches developed so far for the enzymatic production of different Ub chains. These strategies permit a certain flexibility in the production of chains with various linkages and lengths. We critically describe the available methods and comment on advantages and limitations. It is clear that the field is mature to study most of the possible links, but some more work needs to be done to complete the picture and to exploit the current methodologies for understanding in full the Ub code.
authors.affiliation: Department of Pharmacy, University of Parma, 43124 Parma, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Faggiano
authors.affiliation: Maurice Wohl Institute, King's College London, 5 Cutcombe Rd, London SE5 9RX, United Kingdom.
authors.forename: Caterina
authors.initials: C
authors.lastname: Alfano
authors.affiliation: Maurice Wohl Institute, King's College London, 5 Cutcombe Rd, London SE5 9RX, United Kingdom. Electronic address: annalisa.pastore@crick.ac.uk.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Pastore
mesh_terms: D016297:Mutagenesis, Site-Directed; D025821:Polyubiquitin / Q000302:isolation & purification / Q000378:metabolism*; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D025801:Ubiquitin; D025821:Polyubiquitin; D044763:Ubiquitin-Conjugating Enzymes
keywords: Enzymatic ubiquitination; Ligation; Post-translational modification; Protein purification; Ubiquitin; Ubiquitinating enzymes