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| Expression, Purification and Bioassay of Tat-aFGF Fusion Protein in Escherichia coli |
| LIN Jian-cong 1,ZHANG Min-jing1,3 ,SU Zhi-jian1,2,CHEN Hong-xia1,QIU Zhuang-wei1,Lou Guo-feng1, Xiang Qi1,2,3,HUANG Yadong1,2,3 |
1.Life Science and Techonology College of Jinan University，Biopharmaceutical Research & Development Center, Guangzhou 510632,China
2.Guang Dong Provincial Key Laboratory of Bioengineering Medicine，Guangzhou 510632，China
3.National Engineering Research Center of Genetic Medicine，Guangzhou 510632，China |
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Abstract Acidic fibroblast growth factor (aFGF) is a potent neurotrophic factor. It can stimulate the reparation and regeneration of central and peripheral nerves after various injuries. Recently, an approach to deliver therapeutic peptides to the brain is the application of fusion proteins linked to so-called trans-activator transcription (TAT) protein, which can carry the therapeutic protein to permeate blood-brain barrier(BBB) and cell membranes. In this study, aFGF was linked to TAT protein by genetic engineering, and the soluble TAT-aFGF has been expressed successfully in E.Coli. DNA coded fusion proteins Tat-aFGF14-154 and Tat-aFGF27-154 were constructed and cloned into vector pET-3c and the fusion proteins were expressed in E.coli BL21 (DE3). The fusion proteins Tat-aFGF14-154 and Tat-aFGF27-154 were puritified using the combination of CM-Sepharose FF, Heparin affinity chromatography and Sephadex G-25 and the purity were higher than 95%. The fusion proteins were confirmed as Tat-aFGF14-154 and Tat-aFGF27-154 by Western bolt and MALDI-TOF. The mitogenic activity assayed by MTT on Balb/c 3T3 cell showed that Tat-aFGF14-154 and aFGF14-154 had mitogenic activity to Balb/c 3T3 and the best concentration were 1280 ng/ml and 160 ng/ml,respectively.On the other hand, Tat-aFGF27-154 and aFGF27-154 had little mitogenic activity. Tat-aFGF14-154 and Tat-aFGF27-154 could transduce the membrane of PC12, hippocampal neurons, Balb/c 3T3 and HaCaT cells and were mainly located mainly in the cytoplasm detected by immunofluorescence. Four recombinant proteins could efficiently protect hippocampal neurons from toxicity induced by Aβ25-35.
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Received: 22 February 2010
Published: 25 May 2010
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Corresponding Authors:
Ya Dong Huang
E-mail: tydhuang@jnu.edu.cn
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Cite this article:
LIN Jian-Cong, ZHANG Min-Jing, SU Zhi-Jian, CHEN Gong-Xia, QIU Zhuang-Wei, LOU Guo-Feng, XIANG Qi, HUANG E-Dong. Expression, Purification and Bioassay of Tat-aFGF Fusion Protein in Escherichia coli. China Biotechnology, 2010, 30(05): 11-17.
URL:
https://manu60.magtech.com.cn/biotech/ OR https://manu60.magtech.com.cn/biotech/Y2010/V30/I05/11
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| ［1］ Lozano R M , PinedaLucena A, et al. 1H NMR structural characterization of a nonmitogenic, vasodilatory, ischemiaprotector and neuromodulatory acidic fibroblast growth factor. Biochemistry, 2000, 39: 49824993.
［2］ Komi A, Suzuki M, Imamura T. Permeable FGF1 nuclear localization signal peptide stimulates DNA synthesis in various cell types but is celldensity sensitive and unable to support cell proliferation. Exp Cell Res, 1998, 243(2): 408414.
［3］ 余瑛，蔡绍皙，夏玉先. 酸性成纤维细胞生长因子研究进展. 中国药理学通报，2002，18（2）：125128. Yu Y, Cai S H, Xia Y X, et al. Chinese Pharmacological Bulletin, 2002, 18（2）:125128.
［4］ Engele J, Bohn M C. Effects of acidic and basic fibroblast growth factors on glial precursor cell proliferation: age dependency and brain region specificity. Dev Biol, 1992, 152(2): 363372.
［5］ Bannerman P G, Oliver T M, Xu Z, et al. Effects of FGF1 and FGF2 on GD3 immunoreactive spinal neuroepithelial cells. J Neurosci Res, 1996, 45(5): 549557.
［6］ Huang M C, Lo M J, Lin Y L, et al. Functional recovery after the repair of transected cervical roots in the chronic stage of injury. J Neurotrauma, 2009, 26(10): 17951804.
［7］ Huang M C, Chang P T, Tsai M J, et al. Sensory and motor recovery after repairing transected cervical roots. Surg Neurol, 2007, 68 (1): S1724.
［8］ Ricardo B, Maccionil, Cristobal B, et al. Biological markers of Alzheimer'S disease and mild cognitive impairment. Current Alzheimer Research, 2004, 1: 307314.
［9］ Matrone C, Ciotti M T, Mercanti D, et al. NGF and BDNF signaling control amyloidogenic route and Abeta production in hippocampal neurons. Proc Natl Acad Sci U S A, 2008, 105(35): 1313913144.
［10］ Huang T D,Rao Y L,Feng C L, et al. Highlevel expression and purification of TathaFGF19154. Appl Microbiol Biotechnol, 2008, 77:10151022.
［11］ Wang Y, Lin H H, Lin S Q, et al. Cellpenetrating peptide TATmediated delivery of acidic FGF to retina and protection against ischemiareperfusion injury in rats. J. Cell. Mol. Med, 2009(in press).
［12］ 汪小凤，郑青，蔡绍晖，等. 非促有丝分裂型人酸性成纤维细胞生长因子的受体结合特性及对MAPK信号通路的影响. 中国药科大学学报，2005，36（2）：179182 . Wang X F, Zheng Q, Cai S H, et al. Joumal of China Pharmaceutical University. 2005,36(2): 179182.
［13］ Bugatti A, Urbinati C, Ravelli C, et al. Heparinmimicking sulfonic acid polymers as multitarget inhibitors of human immunodeficiency virus type 1 Tat and gp120 proteins. Antimicrob Agents Chemother, 2007, 51(7): 23372345.
［14］ Duchardt F, FotinMleczek M, Schwarz H, et al. A comprehensive model for the cellular uptake of cationic cellpenetrating peptides. Traffic, 2007,8(7):848866.
［15］ Simon M J,Gao S,Kang W H, et al. TATmediated intracellar protein delivery to primary brain cells is dependent on glycosaminoglycan expression. Biotechnol Bioeng, 2009, 104(1): 1019.
［16］ Nakase I, Takeuchi T, Kawabata N, et al. Cellular uptake of argininerich peptides:roles for macropinocytosis and actin rearrangement. Molecular therapy, 2004, 10(6):10111022.
［17］ Wadia J S, Stan R V, Dowdy S F. Transducible TATHA fusogenic peptide enhances escape of TATfusion proteins after lipid raftmacropinocytosis. Nature Medicine, 2004, 10(3):310315.
［18］ Jacob M Gump, Steven F D. TAT transduction: the molecular mechanism and therapeutic prospects. Trends in Molecular Medicine, 2007, 13(10): 443448. |
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