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

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 C 2, 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.