Expression, purification, stability and transduction efficiency of GST-SOD1-R9 fusion protein.

Jianru Pan; Lunqiao WU; Huocong HE; Lijuan Chen; Ying SU; Lingling LI; Shutao Liu

Return

Expression, purification, stability and transduction efficiency of GST-SOD1-R9 fusion protein.

Author: Jianru PAN ¹ ; Lunqiao WU ¹ ; Huocong HE ² ; Lijuan CHEN ¹ ; Ying SU ² ; Lingling LI ¹ ; Shutao LIU ¹
Author Information

1. College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, Fujian, China.
2. 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.
Publication Type:Journal Article
Keywords: GST-SOD1-R9; construction; expression and purification; fusion protein; stability; transduction efficiency
From: Chinese Journal of Biotechnology 2017;33(5):828-837
CountryChina
Language:Chinese
Abstract: 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.