Shanxi Key Laboratory for Modernization of TCVM/ College of Veterinary Medicine, Shanxi Agriculture University, Jinzhong, Shanxi Province, 030800, China.
Shanxi Key Laboratory for Modernization of TCVM/ College of Veterinary Medicine, Shanxi Agriculture University, Jinzhong, Shanxi Province, 030800, China.
Protein Expr Purif. 2024 Sep;221:106519. doi: 10.1016/j.pep.2024.106519. Epub 2024 Jun 1.
Sinopotamon Henanense expresses two metal‒induced metallothioneins (MTs), Cd‒induced MT and Cu‒induced MT (ShCuMT). The Cd‒induced MT has been characterized as a Cd‒thiolate MT. However, it is unknown whether ShCuMT is a Cu‒thiolate MT. In the present study, ShCuMT was expressed heterologously in Escherichia coli and purified by Ni‒NTA column and superdex‒75 column. And its metal‒binding feature was evaluated by DTNB reaction, circular dichroism spectroscopy (CD), isothermal microtitration (ITC), electrospray flight mass spectrometry (ESI‒TOF‒MS), and matrix‒assisted laser desorption ionization flight mass spectrometry (MALDI‒TOF‒MS). Bioinformatics analysis demonstrated that ShCuMT possessed the cysteine‒triplet motif of a Cu‒specific MT. Expression and purification of ShCuMT illustrated that SUMO tag used as the production system for ShCuMT resulted in a high production yield. The stability order of ShCuMT binding metal ions were Cu (Ⅰ) > Cd (Ⅱ) > Zn (Ⅱ). The CD spectrum indicated that ShCuMT binding with Cu (I) exhibited a compact thiol metal clusters structure. Besides, there emerged no a visible nickel‒thiol absorption after Ni‒NTA column affinity chromatography. The ITC results implied that Cu‒ShCuMT possessed the optimal thermodynamic conformation and the highest stoichiometric number of Cu (Ⅰ). Overall, the results suggested that SUMO fusion system is a robust and inexpensive approach for ShCuMT expression and Ni‒NTA column had no influence on metal binding of ShCuMT and Cu(Ⅰ) was considered its cognate metal ion, and ShCuMT possessed canonical Cu‒thiolate characteristics. The metal binding feature of ShCuMT reported here contributes to elucidating the structure‒function relationship of ShCuMT in S. Henanense.
河南华溪蟹表达两种金属诱导金属硫蛋白（MTs），Cd 诱导 MT 和 Cu 诱导 MT（ShCuMT）。Cd 诱导 MT 已被鉴定为 Cd-硫醇 MT。然而，ShCuMT 是否是 Cu-硫醇 MT 尚不清楚。在本研究中，ShCuMT 在大肠杆菌中异源表达并通过 Ni-NTA 柱和 superdex-75 柱纯化。通过 DTNB 反应、圆二色光谱（CD）、等温微量滴定（ITC）、电喷雾飞行质谱（ESI-TOF-MS）和基质辅助激光解吸电离飞行质谱（MALDI-TOF-MS）评估其金属结合特性。生物信息学分析表明，ShCuMT 具有 Cu 特异性 MT 的半胱氨酸三联体基序。ShCuMT 的表达和纯化表明，SUMO 标签作为 ShCuMT 的生产系统可获得高产率。ShCuMT 结合金属离子的稳定性顺序为 Cu（Ⅰ）>Cd（Ⅱ）>Zn（Ⅱ）。CD 光谱表明，ShCuMT 与 Cu（I）结合表现出紧凑的硫醇金属簇结构。此外，Ni-NTA 柱亲和层析后没有出现可见的镍-硫醇吸收。ITC 结果表明，Cu-ShCuMT 具有最佳热力学构象和最高的 Cu（Ⅰ）计量数。总体而言，结果表明 SUMO 融合系统是 ShCuMT 表达的一种强大且廉价的方法，Ni-NTA 柱对 ShCuMT 金属结合没有影响，Cu（Ⅰ）被认为是其同源金属离子，ShCuMT 具有典型的 Cu-硫醇特征。这里报道的 ShCuMT 的金属结合特性有助于阐明 S. Henanense 中 ShCuMT 的结构-功能关系。
