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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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