title: Preparation of distinct ubiquitin chain reagents of high purity and yield.
abstract: The complexity of protein ubiquitination signals derives largely from the variety of polyubiquitin linkage types that can modify a target protein, each imparting distinct functional consequences. Free ubiquitin chains of uniform linkages and length are important tools in understanding how ubiquitin-binding proteins specifically recognize these different polyubiquitin modifications. While some free ubiquitin chain species are commercially available, mutational analyses and labeling schemes are limited to select, marketed stocks. Furthermore, the multimilligram quantities of material required for detailed biophysical and/or structural studies often makes these reagents cost prohibitive. To address these limitations, we have optimized known methods for the synthesis and purification of linear, K11-, K48-, and K63-linked ubiquitin dimers, trimers, and tetramers on a preparative scale. The high purity and relatively high yield of these proteins readily enables material-intensive experiments and provides flexibility for engineering specialized ubiquitin chain reagents, such as fluorescently labeled chains of discrete lengths.
authors.affiliation: Department of Structural Biology, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Ken C
authors.initials: KC
authors.lastname: Dong
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Helgason
authors.forename: Christine
authors.initials: C
authors.lastname: Yu
authors.forename: Lilian
authors.initials: L
authors.lastname: Phu
authors.forename: David P
authors.initials: DP
authors.lastname: Arnott
authors.forename: Ivan
authors.initials: I
authors.lastname: Bosanac
authors.forename: Deanne M
authors.initials: DM
authors.lastname: Compaan
authors.forename: Oscar W
authors.initials: OW
authors.lastname: Huang
authors.forename: Anna V
authors.initials: AV
authors.lastname: Fedorova
authors.forename: Donald S
authors.initials: DS
authors.lastname: Kirkpatrick
authors.forename: Sarah G
authors.initials: SG
authors.lastname: Hymowitz
authors.forename: Erin C
authors.initials: EC
authors.lastname: Dueber
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D005822:Genetic Vectors; D025821:Polyubiquitin / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015202:Protein Engineering / Q000379:methods*; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D025801:Ubiquitin / Q000096:biosynthesis / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D025801:Ubiquitin; D025821:Polyubiquitin