Cloning, Expression, and Characterization of a Recombinant Gilthead Seabream Growth Hormone

Abstract

cDNA clones coding for the gilthead seabream (Sparus aurata) growth hormone (sbGH) were isolated from a pituitary expression library using a flounder cDNA probe. The nucleotide sequence of a GH cDNA clone containing an insert of 896 nucleotides was determined. The cDNA encoded a polypeptide of 204 amino acids including a signal peptide of 17 amino acids and contained a 5′ and a 3′ untranslated region of 48 and 233 nucleotides; respectively. The mRNA determined by Northern blot was approximately 1 kb. Amino acid sequence homologies of 97.1% with red seabream GH, 88.9% with the tuna GH, and 67% with the coho salmon GH was found. Transient expression of a sbGH cDNA was done in HeLa cells by induction with a vaccinia virus system, and the expressed GH was detected by immunofluorescence and immunoprecipitation with a specific antibody to the native sbGH. The sbGH cDNA was expressed in Escherichia coli by using the pGEX-3X and the pET-3a expression systems. The recombinant sbGH expressed in the pET-3a system was similar, if not identical, to the native hormone when analyzed by homologous radioimmunoassay and receptor binding assay.