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Zebrafish eggs used as bioreactors for the production of bioactive tilapia insulin-like growth factors

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Abstract

Multiple advantages-including the short generation time, large numbers of fertilized eggs, low cost of cultivation and easy maintenance favor the use of fish as bioreactors for the production of pharmaceutical proteins. In the present study, zebrafish eggs were used as bioreactors to produce mature tilapia insulin-like growth factors (IGFs) proteins using the oocyte-specific zona pellucida (zp3) promoter. The chimeric expression plasmids, pT2-ZP-tIGFs-IRES-hrGFP, in which hrGFP was used as reporter of tilapia IGFs expression, were designed to established Tg (ZP:tIGFs:hrGFP) transgenic lines for the expression of tilapia IGF-1 and IGF-2. Recombinant tilapia IGF-1 and IGF-2 were expressed as soluble forms in cytoplasm of fertilized eggs. The content level of tilapia IGF-1 and IGF-2 were 6.5 and 5.0% of the soluble protein, respectively. Using a simple Ni–NTA affinity chromatography purification process, 0.58 and 0.49 mg of purified tilapia IGF-1 and IGF-2 were obtained, respectively, from 650 fertilized eggs. The biological activity of the purified tilapia IGF-1 and IGF-2 was confirmed via a colorimetric bioassay to monitor the growth stimulation of zebrafish embryonic cells (ZF4), tilapia ovary cells (TO-2) and human osteosarcoma epithelial cells (U2OS). These results demonstrate that the use of zebrafish eggs as bioreactors is a promising approach for the production of biological recombinant proteins.

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Acknowledgments

Mass spectrometry analyses were performed by the Core Facilities for Proteomics Research located at the Institute of Biological Chemistry, Academia Sinica, supported by a National Science Council grant (NSC 94-3112-P-001-010-Y) and the Academia Sinica. We would like to thank our lab colleagues for their constructive criticism and helpful suggestions. This work was supported by grants from the Academia Sinica, Taiwan (94S-1402) and the National Science Council NSC 97-2317-B-001-012-CC1.

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Authors and Affiliations

  1. Laboratory of Marine Molecular Biology and Biotechnology 301, Institute of Cellular and Organismic Biology, Academia Sinica, No.128, Section 2, Academia Road, Nankang, Taipei, 115, Taiwan

    Shao-Yang Hu, Chia-Hsuan Liao, Yi-Pei Lin, Yen-Hsing Li, Gen-Hwa Lin, Tzu-Hsuan Yang & Jen-Leih Wu

  2. Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan

    Hong-Yi Gong

  3. Division of Molecular and Developmental Biology, National Institute of Genetics, Mishima, Shizuoka, Japan

    Koichi Kawakami

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  1. Shao-Yang Hu
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  2. Chia-Hsuan Liao
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  3. Yi-Pei Lin
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  4. Yen-Hsing Li
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  9. Jen-Leih Wu
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Correspondence to Jen-Leih Wu.

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Hu, SY., Liao, CH., Lin, YP. et al. Zebrafish eggs used as bioreactors for the production of bioactive tilapia insulin-like growth factors. Transgenic Res 20, 73–83 (2011). https://doi.org/10.1007/s11248-010-9388-3

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  • DOI: https://doi.org/10.1007/s11248-010-9388-3

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