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

Citation:

PDF (1668 KB)

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

More Information

Graphical Abstract

Abstract

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

FullText(HTML)

References (21)

References

[1]	Kharitonenkov A, Adams AC. Inventing new medicines: the FGF21 story[J].Mol Metab,2014,3(3):221-229.
[2]	Degirolamo C,Sabba C,Moschetta A.Therapeutic potential of the endocrine fibroblast growth factors FGF19,FGF21 and FGF23[J].Nat Rev Drug Discov,2015,15(1):51-69.
[3]	Bookout AL, De Groot MH, Owen BM, et al. FGF21 regulates metabolism and circadian behavior by acting on the nervous system[J].Nat Med,2013,19(9):1147-1152.
[4]	Sa-Nguanmoo P,Chattipakorn N,Chattipakorn SC.Potential roles of fibroblast growth factor 21 in the brain[J].Metab Brain Dis,2016,31(2):239-248.
[5]	Hsuchou H,Pan W,Kastin AJ.The fasting polypeptide FGF21 can enter brain from blood[J].Peptides,2007,28(12):2382-2386.
[6]	Kristensen M,Birch D,Nielsen HM.Applications and challenges for use of cell-penetrating peptides as delivery vectors for peptide and protein cargos[J].Int J Mol Sci,2016,17(2):185.
[7]	El-S K,Morishita M,Kamei N,et al.Efficiency of cell-penetrating peptides on the nasal and intestinal absorption of therapeutic peptides and proteins[J].Int J Pharm,2009,381(1):49-55.
[8]	Kamei N,Takeda-Morishita M.Brain delivery of insulin boosted by intranasal coadministration with cell-penetrating peptides[J].J Control Release,2015,197:105-110.
[9]	Rizzuti M,Nizzardo M,Zanetta C,et al.Therapeutic applications of the cell-penetrating HIV-1 Tat peptide[J].Drug Discov Today,2015,20(1):76-85.
[10]	Meloni BP,Milani D,Edwards AB,et al.Neuroprotective peptides fused to arginine-rich cell penetrating peptides:neuroprotective mechanism likely mediated by peptide endocytic properties[J].Pharmacol Ther,2015,153:36-54.
[11]	Herce HD,Garcia AE,Litt J,et al.Arginine-rich peptides destabilize the plasma membrane,consistent with a pore formation translocation mechanism of cell-penetrating peptides[J].Biophys J,2009,97(7):1917-1925.
[12]	Yin W,Cao G,Johnnides MJ,et al.TAT-mediated delivery of Bcl-xL protein is neuroprotective against neonatal hypoxic-ischemic brain injury via inhibition of caspases and AIF[J].Neurobiol Dis,2006,21(2):358-371.
[13]	Namikoshi A,Wu JL,Yamashita T,et al.Vaccination trials with Penaeus japonicus to induce resistance to white spot syndrome virus[J].Aquaculture,2004,229(1/2/3/4):25-35.
[14]	Yu HY,Chen S,Xu Z,et al.Protective effect of fibroin peptides on Aβ_25-35-induced injury in SH-SY5Y cells and its mechanism[J].J China Pharm Univ,2017,48(5):609-613.
[15]	Ishii M,Iadecola C.Metabolic and non-cognitive manifestations of Alzheimer′s disease:the hypothalamus as both culprit and target of pathology[J].Cell Metab,2015,22(5):761-776.
[16]	Higuchi M,Maeda J,Ji B,et al.In-vivovisualization of key molecular processes involved in Alzheimer′s disease pathogenesis:Insights from neuroimaging research in humans and rodent models[J].BBA-Mol Basis Dis,2010,1802(4):373-388.
[17]	Anand A,Patience AA,Sharma N,et al.The present and future of pharmacotherapy of Alzheimer′s disease:a comprehensive review[J].Eur J Pharmacol,2017,815:364-375.
[18]	Guo JJ,Liao H.Development of drug for Alzheimer′s disease[J].J China Pharm Univ(中国药科大学学报),2010,41(5):395-400.
[19]	Sanguanmoo P,Tanajak P,Kerdphoo S,et al.FGF21 improves cognition by restored synaptic plasticity,dendritic spine density,brain mitochondrial function and cell apoptosis in obese-insulin resistant male rats[J].Horm Behav,2016,85:86-95.
[20]	Mariani E,Polidori MC,Cherubini A,et al.Oxidative stress in brain aging,neurodegenerative and vascular diseases:an overview[J].J Chromatogr B,2005,827(1):65-75.
[21]	Bhat AH,Dar KB,Anees S,et al.Oxidative stress,mitochondrial dysfunction and neurodegenerative diseases:a mechanistic insight[J].Biomed Pharmacother,2015,74:101-110.

Cited By

Get Citation

PDF

XML

Article views (1269) PDF downloads (1427)

Turn off MathJax

Article Contents

Abstract

References

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

Abstract

References

Catalog

Export File

Citation

Format

Content