title: Construction of a mouse Aos1-Uba2 chimeric SUMO-E1 enzyme, mAU, and its expression in baculovirus-insect cells.
abstract: Small ubiquitin-related modifier (SUMO) is a highly conserved protein that is covalently attached to target proteins. This posttranslational modification, designated SUMOylation, is a major protein-conjugation-driven strategy designed to regulate structure and function of cellular proteins. SUMOylation consists of an enzymatic cascade involving the E1-activating enzyme and the E2-conjugating enzyme. The SUMO-E1 enzyme consists of two subunits, a heterodimer of activation of Smt3p 1 (Aos1) and ubiquitin activating enzyme 2 (Uba2), which resembles the N- and C-terminal halves of ubiquitin E1 (Uba1). Herein, we describe the rational design of a single polypeptide version of SUMO-E1, a chimera of mouse Aos1 and Uba2 subunits, termed mAU, in which the functional domains appear to be arranged in a fashion similar to Uba1. We also describe the construction of a mAU plasmid for expression in a baculovirus-insect cell system and present an in situ SUMOylation assay using the recombinant mAU. Our results showed that mAU has SUMO-E1 activity, thereby indicating that mAU can be expressed in baculovirus-insect cells and represents a suitable source of SUMO-E1.
authors.affiliation: Department of Biological Sciences; Graduate School of Science and Technology; Kumamoto University; Kumamoto, Japan.
authors.forename: Tomofumi
authors.initials: T
authors.lastname: Nakayama
authors.affiliation: Department of Biological Sciences; Graduate School of Science and Technology; Kumamoto University; Kumamoto, Japan.
authors.forename: Eri
authors.initials: E
authors.lastname: Yuasa
authors.affiliation: Department of Biological Sciences; Graduate School of Science and Technology; Kumamoto University; Kumamoto, Japan.
authors.forename: Ayumi
authors.initials: A
authors.lastname: Kanemaru
authors.affiliation: Department of Biological Sciences; Graduate School of Science and Technology; Kumamoto University; Kumamoto, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Saito
authors.affiliation: Department of Biological Sciences; Graduate School of Science and Technology; Kumamoto University; Kumamoto, Japan; Department of New Frontier Sciences; Graduate School of Science and Technology; Kumamoto University; Kumamoto, Japan.
authors.forename: Hisato
authors.initials: H
authors.lastname: Saitoh
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D051379:Mice; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D061987:Sf9 Cells; D018411:Spodoptera / Q000502:physiology; D014161:Transduction, Genetic / Q000379:methods*; D044764:Ubiquitin-Activating Enzymes / Q000235:genetics* / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D044764:Ubiquitin-Activating Enzymes
keywords: E1 enzyme; baculovirus; chimera-fusion protein; posttranslational modification; protein expression; protein structure; small ubiquitin-related modifier (SUMO); ubiquitin