Pan Jianru, Wu Lunqiao, He Huocong, Chen Lijuan, Su Ying, Li Lingling, Liu Shutao
College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, Fujian, China.
Fujian Cancer Hospital & Fujian Medical University Cancer Hospital, Laboratory of Radiation Oncology and Radiobiology, Fujian Key Laboratory of Tumor Translational Cancer Medicine, Fuzhou 350014, Fujian, China.
Sheng Wu Gong Cheng Xue Bao. 2017 May 25;33(5):828-837. doi: 10.13345/j.cjb.160401.
The fusion of cell permeable peptide TAT and bifunctional antioxidant enzymes, GST (Glutathione sulfur transferase)-TAT-SOD1 (Cu, Zn superoxide dismutase), is an intracellular superoxide scavenger. Compared with SOD1-TAT, GST-TAT-SOD1 has better protective effect on oxidative damage but less transduction efficiency. A novel cell permeable bifunctional antioxidant enzymes with the fusion of GST, SOD1 and polyarginine R9 was constructed for higher transduction efficiency. The full nucleotide sequence of SOD1-R9 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1 with the GST tag. After the successful construction of the prokaryotic expression vectors of GST-SOD1-R9, the recombinant vector was then transformed into Escherichia coli BL21 (DE3) and the GST-SOD1-R9 fusion protein was produced with the induction of IPTG. The soluble expression of GST-SOD1-R9 fusion protein was combining with the induction temperature and time. The best soluble expression was obtained with the induction temperature of 25 ℃ and the induction time of 11 h. The fusion protein was purified through the combination of 80% ammonium sulfate precipitation and affinity chromatography using glutathione agarose, and verified by SDS-PAGE and special enzymatic activity. The thermal and pH stability of GST-SOD1-R9 fusion protein were analyzed and the SOD and GST activity of fusion protein were proved to be well maintained under physiological conditions. Finally, the transduction efficiency of GST-SOD1-R9 fusion protein was proved to be better than GST-TAT-SOD1 fusion protein (P<0.05). These works establish a foundation for further study of the protective effect of GST-SOD1-R9 fusion protein against oxidative damage.
细胞穿透肽TAT与双功能抗氧化酶谷胱甘肽硫转移酶（GST）-TAT-超氧化物歧化酶1（Cu, Zn超氧化物歧化酶，SOD1）的融合体是一种细胞内超氧化物清除剂。与SOD1-TAT相比，GST-TAT-SOD1对氧化损伤具有更好的保护作用，但转导效率较低。构建了一种新型的由GST、SOD1和多聚精氨酸R9融合而成的细胞穿透双功能抗氧化酶，以提高转导效率。合成了SOD1-R9的全长核苷酸序列，并将其插入带有GST标签的原核表达载体pGEX-4T-1中。成功构建GST-SOD1-R9的原核表达载体后，将重组载体转化到大肠杆菌BL21（DE3）中，并用异丙基-β-D-硫代半乳糖苷（IPTG）诱导产生GST-SOD1-R9融合蛋白。GST-SOD1-R9融合蛋白的可溶性表达与诱导温度和时间有关。在诱导温度为25℃、诱导时间为11 h时获得最佳可溶性表达。通过80%硫酸铵沉淀和使用谷胱甘肽琼脂糖的亲和层析相结合的方法纯化融合蛋白，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和特殊酶活性进行验证。分析了GST-SOD1-R9融合蛋白的热稳定性和pH稳定性，结果表明融合蛋白的超氧化物歧化酶（SOD）和谷胱甘肽硫转移酶（GST）活性在生理条件下得到了很好的维持。最后，证明GST-SOD1-R9融合蛋白的转导效率优于GST-TAT-SOD1融合蛋白（P<0.05）。这些工作为进一步研究GST-SOD1-R9融合蛋白对氧化损伤的保护作用奠定了基础。
