Construction, expression, purification and neuroprotective activity of TAT-FGF21 fusion protein

CHEN Suting; CHEN Song; GAO Xiangdong

doi:10.11665/j.issn.1000-5048.20180417

Journal of China Pharmaceutical University > 2018 > 49(4): 496-501. > DOI: 10.11665/j.issn.1000-5048.20180417

CHEN Suting, CHEN Song, GAO Xiangdong. Construction, expression, purification and neuroprotective activity of TAT-FGF21 fusion protein[J]. Journal of China Pharmaceutical University, 2018, 49(4): 496-501. DOI: 10.11665/j.issn.1000-5048.20180417

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Construction, expression, purification and neuroprotective activity of TAT-FGF21 fusion protein

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Abstract

In order to improve the brain distribution of fibroblast growth factor 21(FGF21), TAT-FGF21 fusion protein was designed and its neuroprotective activity was investigated. The recombinant plasmid of pET28a-TAT-FGF21 was constructed and transformed into E. coli BL-21(DE3)sensitive bacteria. The TAT-FGF21 fusion protein was purified by Ni-NTA affinity chromatography column after IPTG induced expression. The SH-SY5Y cell damage model was induced by Aβ_25-35, and the TAT-FGF21 fusion protein was used to intervene. The effects of Aβ_25-35 and TAT-FGF21 induced on SH-SY5Y cell viability were determined using MTT method; DCFH-DA fluorescent probe was used to detect the intervention effect TAT-FGF21 on reactive oxygen species(ROS)generation induced by Aβ_25-35 in SH-SY5Y cells; the effects of Aβ_25-35 and TAT-FGF21 on mitochondrial membrane potential in SH-SY5Y cells were detected with JC-1 fluorescent probe. The results showed that TAT-FGF21 could improve the viability of SH-SY5Y cells, reduce the intracellular ROS production level of SH-SY5Y cells, and enhance the mitochondrial membrane potential of SH-SY5Y cells, which indicate that TAT-FGF21 could protect neurons on SH-SY5Y cell injury induced by Aβ_25-35 through alleviating oxidative damage.
- TAT-FGF21,
- expression,
- purification,
- neuroprotective activity,
- reactive oxygen species,
- mitochondrial membrane potential

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