title: Zebrafish eggs used as bioreactors for the production of bioactive tilapia insulin-like growth factors.
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.
authors.affiliation: 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.
authors.forename: Shao-Yang
authors.initials: SY
authors.lastname: Hu
authors.forename: Chia-Hsuan
authors.initials: CH
authors.lastname: Liao
authors.forename: Yi-Pei
authors.initials: YP
authors.lastname: Lin
authors.forename: Yen-Hsing
authors.initials: YH
authors.lastname: Li
authors.forename: Hong-Yi
authors.initials: HY
authors.lastname: Gong
authors.forename: Gen-Hwa
authors.initials: GH
authors.lastname: Lin
authors.forename: Koichi
authors.initials: K
authors.lastname: Kawakami
authors.forename: Tzu-Hsuan
authors.initials: TH
authors.lastname: Yang
authors.forename: Jen-Leih
authors.initials: JL
authors.lastname: Wu
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D019149:Bioreactors; D002460:Cell Line; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis / Q000737:chemistry; D007335:Insulin-Like Growth Factor II / Q000096:biosynthesis / Q000737:chemistry; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D010063:Ovum / Q000166:cytology / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D013002:Somatomedins / Q000096:biosynthesis* / Q000737:chemistry; D017210:Tilapia / Q000235:genetics / Q000378:metabolism*; D015027:Zebrafish / Q000235:genetics* / Q000254:growth & development; D015044:Zona Pellucida / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II