Prokaryotic Expression, Purification and Deubiquitinase Activity Assay of TaWTG1 in Wheat (Triticum aestivum)
ZHANG Wen-Jing1, CHEN Hai-Chao1, GUO Li-Jian1, LIU Xiang-Li1,2,*, ZHAO Hui-Xian1,2,*
1 College of Life Science, Northwest A&F University, Yangling 712100, China; 2 State Key Labaratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling 712100, China;
Abstract：Deubiquitinating enzyme is a reverse modulating enzyme of ubiquitination pathway and is involved in the regulation of protein degradation. In order to explore the gene function of deubiquitinating enzyme family member WTG1 (wide and thick grain 1) in wheat (Triticum aestivum), the physical and chemical properties of TaWTG1 encoded protein as well as structure were analyzed by bioinformatics method in this study. The prokaryotic expression vector pET28a-TaWTG1 was constructed and transformed into Escherichia coli expression strain BL21(DE3). The induced-expression conditions, including culture temperature, isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration and induction time, were optimized. The solubility of recombinant protein His-TaWTG1 was analyzed, and its deubiquitination activity was verified in vitro after purified by nickel column. The results showed that the TaWTG1 protein consists of 319 amino acid residues with a theoretical molecular weight of 35.38 kD and an isoelectric point of 4.62. The tertiary structure of the protein is mainly composed of α-helix, which contains the otubain conserved domain of the ovarian tumor-related proteases (OTU)-related protease family. The optimal induced expression conditions of the recombinant protein His-TaWTG1 were 0.4 mmol/L IPTG at 28 °C for 6 h. The recombinant protein His-TaWTG1 mainly existed in supernatant in compatible form. Western blot analysis identified the recombinant protein purified by nickel column as the target protein. In vitro activity assay showed that TaWTG1 had deubiquitination enzyme activity and could cleave lysine (K) 48 and K63-linked tetrameric ubiquitin chains. These results provide general data for further functional study of TaWTG1 gene in wheat.
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