College of Pharmacy, Jinan University, Guangzhou 510632, China.
Protein J. 2013 Apr;32(4):259-65. doi: 10.1007/s10930-013-9482-5.
In this study, a novel iron superoxide dismutase (FeSOD) gene from Sonneratia alba was cloned and then expressed in Escherichia coli Rosetta-gami, designated as SaFeSOD. The DNA sequence of SaFeSOD contained a 786-bp open reading frame which encodes a 261 amino-acid protein of 30.0 kDa. The 651-bp fragment coding for putative mature SaFeSOD was amplified and inserted into pET15b for expression. This recombinant SaFeSOD was subsequently isolated by Ni-trap column protein purification system. The apparent molecular mass of the purified enzyme was 25 kDa on SDS-PAGE. In comparison with FeSODs from other plant species, all iron-binding sites (His 27, His 80, Asp 164 and His 168) of SaFeSOD were conserved. SaFeSOD was found to have good pH stability in the pH range of 3.5-9.5 at 25 °C after 1 h incubation and was relatively stable and showed 78 % activity when incubated in 50 °C for 1 h. Quantitative real-time PCR experiments demonstrated that SaFeSOD was expressed in leaf, stem, flower, fruit and root tissues with the highest expression in leaf tissues.
在这项研究中，从白骨壤中克隆了一种新型的铁超氧化物歧化酶（FeSOD）基因，并在大肠杆菌 Rosetta-gami 中表达，命名为 SaFeSOD。SaFeSOD 的 DNA 序列包含一个 786bp 的开放阅读框，编码一个 261 个氨基酸的 30.0kDa 蛋白质。编码假定成熟 SaFeSOD 的 651bp 片段被扩增并插入 pET15b 中进行表达。通过 Ni-亲和层析柱蛋白纯化系统分离出这种重组 SaFeSOD。纯化酶的表观分子量在 SDS-PAGE 上为 25kDa。与其他植物物种的 FeSOD 相比，SaFeSOD 的所有铁结合位点（His 27、His 80、Asp 164 和 His 168）均保守。SaFeSOD 在 25°C 下孵育 1 小时时，在 pH3.5-9.5 的范围内具有良好的 pH 稳定性，在 50°C 下孵育 1 小时时相对稳定，活性为 78%。实时定量 PCR 实验表明，SaFeSOD 在叶片、茎、花、果实和根组织中表达，在叶片组织中表达量最高。
