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High-level expression and purification of Tat-haFGF19-154

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Abstract

Human acidic fibroblast growth factor (haFGF) stimulates repair and regeneration of central and peripheral nerves after various injuries. However, it is unable to cross the blood–brain barrier (BBB). To produce a therapeutic haFGF with cell-permeable activity, we fused the haFGF19-154 gene with Tat-PTD. After its construction by a single-step insertion of a polymerase chain reaction (PCR)–amplified coding sequence, the vector pTat-haFGF19-154-His was expressed in Escherichia coli BL21 (DE3) cells. The optimal expression level of the soluble fusion protein was up to 36.7% of the total cellular protein. The recombinant Tat-haFGF19-154-His was purified by a combination of Ni–NTA affinity, Sephadex G-25, and heparin affinity chromatography to 95% as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The final yield was 171 mg/l culture. Purified Tat-haFGF19-154-His had distinct mitogenic activity in Balb/c 3T3 cells, as measured by methylthiazoletetrazolium (MTT) assay and its ED50 was 3.931 × 10−4 µmol/l. Tat-haFGF19-154-His protein intravenously injected at the dose of 10 mg/kg could be detected in the pallium and hippocampi.

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Acknowledgment

We are grateful to Dr. Xionglei He for helpful comments on this manuscript. This project was supported in part by grants from the National High-Tech Research and Development Program of China (2002AA2Z3318 and 2001AA215131), National Natural Science Foundation (30670541), National Basic Research Priorities Program (G1999054204), the Program of New Century Excellent Talents in University (X. Li), Zhejiang Provincial Program for Innovative Scientists in Health Sciences (X. Li) and Zhejiang Provincial Natural Science Foundation (Z205755)

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Authors and Affiliations

  1. Biopharmaceutical Research & Development Center, Jinan University College of Pharmacy, Guangzhou, Guangdong, 510632, People’s Republic of China

    Yadong Huang, Yanmei Li, Xiaoping Wu & Zhijian Su

  2. School of Pharmaceutical Science, Wenzhou Medical College, Wenzhou, Zhejiang, 325035, People’s Republic of China

    Yulan Rao, Jian Xiao & Xiaokun Li

  3. Center for Instrumental Analysis, Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, Nanjing, Jiangsu, 210009, People’s Republic of China

    Yulan Rao

  4. Shaanxi Institute of Zoology, Xi’an, Shaanxi, 710032, People’s Republic of China

    Chengli Feng

  5. Engineering Research Center of Bioreactor and Pharmaceutical Development, Ministry of Education, Jilin Agricultural University, Changchun, Jilin, 130118, People’s Republic of China

    Yechen Xiao & Xiaokun Li

  6. Department of Medicine, University of Louisville, Louisville, KY, 40292, USA

    Wenke Feng

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  1. Yadong Huang
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  2. Yulan Rao
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  9. Wenke Feng
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Correspondence to Wenke Feng or Xiaokun Li.

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Yadong Huang and Yulan Rao contributed equally to this work.

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Huang, Y., Rao, Y., Feng, C. et al. High-level expression and purification of Tat-haFGF19-154 . Appl Microbiol Biotechnol 77, 1015–1022 (2008). https://doi.org/10.1007/s00253-007-1249-5

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  • DOI: https://doi.org/10.1007/s00253-007-1249-5

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