Expression and activity of single‐chain recombinant eel follicle stimulating hormone in mammalian cells (792.2)

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The glycoprotein hormone family consists of luteinizing hormone (LH), follicle‐stimulating hormone (FSH), and thyroid‐stimulating hormone (TSH), which are secreted by the pituitary gland in all mammalian species, and chorionic gonadotropin (CG), which is secreted by placental trophoblast cells in primates and equids. These hormones consists of non‐covalently associated α and β subunits. Within a species, the amino acid sequence of the α subunit is identical across all glycoprotein hormones and is encoded by a single gene. The αβ dimer is the active form of the hormone, and biological specificity is conferred by the β subunit. Also in fish, the duality of gonadotropin hormone (GTH), GTH‐1 (FSH‐like GTH), and GTH‐II (LH‐like GTH) is found in certain teleost. In this study, in order to understand the fundamental mechanisms involved in teleost reproduction and to proved a broader basis for comparative studt of teleost GTHs, we isolated a cDNA encoding the α and β subunit of GHT‐1 from eel and produced it's recombinant protein from E.coli and CHO cells. To obtain tethered eelFSH, the cDNA encoding the full‐length eel FSH β‐subunit (signal sequence of 22 amino acid residues and the mature protein of 105 amino acid residues) was fused with the mature protein (93 amino acids) of the α‐subunit by overlapping PCR mutagenesis. Subsequently, PCR fragement was inserted into the pCR2.1 clon ing vector and sequenced. After then, it was digested with the EcoRI and SalI enzymes and ligated into the eukaryotic expression vector pcDNA3. The gene was completely sequenced to confirm the presence of the Kozak site and, myc‐tag and to role out the possibility of any PCR erros. Eel FSH protein was expressed in the E. coli system using the pREST expression plasmid vector, and the protein was purified by NI‐NTA column. Eel FSH protein was detected at the predicted molecular weight of 24.3 kDa by SDS‐PAGE in E.coli and 32‐45 kDa in CHO cell lines.Grant Funding Source: This work was supported by NFRDI