重金属相关异戊二烯化植物蛋白（HIPPs）由于其独特的重金属结合域和异戊二烯序列的结构特点,成为一类重要的金属分子伴侣。为鉴定茶树（Camellia sinensis）黄金芽CsHIPP26.1蛋白的螯合离子,将pET-32a-CsHIPP26.1重组质粒和空载体分别转入大肠杆菌BL21,在分别添加4 mol·L-1的单一金属离子（CuCl2、ZnCl2、MgCl2、FeCl3、CaCl2）或5种金属离子混合液以及1 mmol·L-1 IPTG诱导的LB液体中培养,观测大肠杆菌的生长情况,并用His-tag蛋白纯化磁珠纯化获得融合目的蛋白,经原子吸收分光光度计分析融合蛋白中金属离子含量,计算蛋白螯合的离子数目。结果表明,CsHIPP26.1蛋白仅与Zn2+和Cu2+螯合,且螯合Zn2+的能力显著强于Cu2+。根据其结合金属离子与目的蛋白质量摩尔比推测,CsHIPP26.1蛋白螯合Zn2+、Cu2+金属离子的最大数目分别为2和1。
Heavy metal-associated isoprenylated plant proteins (HIPPs) is an important metallochaperones due to its unique heavy metal binding domains (HMA) and the structural characteristics of isoprenylation motif. In order to identify the chelating ions of CsHIPP26.1 protein in Camellia sinensis (L.) cv. ‘Huangjinya’, the pET-32a-CsHIPP26.1 recombinant plasmids and empty carriers were respectively transferred into E. coli BL21, and then were cultured in LB liquid culture medium with 4 mol·L-1 single metal ions (CuCl2, ZnCl2, MgCl2, FeCl3, CaCl2) or multiple metal ions and 1 mmol·L-1 IPTG. The growth of E. coli in different ion media was observed, meanwhile the fusion target protein was obtained by His-tag protein purification magnetic bead. The contents of metal ions in fusion protein were analyzed by atomic absorption spectrophotometer, and the number of ions chelated by the protein was calculated. The results show that CsHIPP26.1 protein was only chelated with Zn2+ and Cu2+, and the chelating ability to Zn2+ was significantly higher than Cu2+. Based on the molar ratio of its bound metal ions to the target protein, the maximum number of Zn2+, Cu2+ chelated by CsHIPP26.1 protein was 2 and 1, respectively.
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