title: The expression, purification and crystallization of a ubiquitin-conjugating enzyme E2 from Agrocybe aegerita underscore the impact of His-tag location on recombinant protein properties.
abstract: Ubiquitination is a post-translational modification involved in myriad cell regulation and disease pathways. The ubiquitin-conjugating (E2) enzyme is the central player in the ubiquitin-transfer pathway. Although a large array of E2 structures are available, not all E2 families have known structures and three-dimensional structures from fungal organisms other than yeast are lacking. Here, the expression, purification, crystallization and preliminary X-ray analysis of UbcA1, a novel ubiquitin-conjugating enzyme identified from the medicinal mushroom Agrocybe aegerita, which shows antitumour properties, are reported. As a potential anticancer drug candidate, the protein was expressed in either a C-terminally or an N-terminally His-tagged form. In the process of purification and crystallization, the location of the His tag seemed to play a crucial role in protein stability. In contrast to unsuccessful crystallization trials for the protein with a C-terminal tag, a crystal of N-terminally His-tagged UbcA1 grown under optimal conditions diffracted X-rays to 1.7 Å resolution. The crystal belonged to space group C2, with unit-cell parameters a = 84.93, b = 34.76, c = 128.10 Å, β = 118.57°. An X-ray data set was collected that was suitable for structure determination, showing satisfactory completeness, <I/σ(I)> and R factors. All of these results underscore the non-negligible impact of His-tag location on protein behaviour during the process of purification and crystallization.
authors: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, People's Republic of China.
authors: De-Feng
authors: DF
authors: Li
authors: Lei
authors: L
authors: Feng
authors: Yan-Jie
authors: YJ
authors: Hou
authors: Wei
authors: W
authors: Liu
mesh_terms: D055362:Agrocybe / Q000201:enzymology*; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine; D044763:Ubiquitin-Conjugating Enzymes
keywords: Agrocybe aegerita; UbcA1; ubiquitin; ubiquitin-conjugating enzyme E2
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