CLONING, EXPRESSION, AND CHARACTERIZATION OF A RECOMBINANT GILTHEAD SEABREAM GROWTH-HORMONE

cDNA clones coding for the gilthead seabream (Sparus aurata) growth ho rmone (sbGH) were isolated from a pituitary expression library using a flounder cDNA probe. The nucleotide sequence of a GH cDNA clone conta ining 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 nu cleotides, respectively. The mRNA determined by Northern blot was appr oximately 1 kb. Amino acid sequence homologies of 97.1% with red seabr eam GH, 88.9% with the tuna GH, and 67% with the coho salmon GH was fo und. Transient expression of a sbGH cDNA was done in HeLa cells by ind uction with a vaccinia virus system, and the expressed GH was detected by immunofluorescence and immunoprecipitation with a specific antibod y to the native sbGH. The sbGH cDNA was expressed in Escherichia coli by using the pGEX-3X and the pET-3a expression systems. The recombinan t sbGH expressed in the pET-3a system was similar, if not identical, t o the native hormone when analyzed by homologous radioimmunoassay and receptor binding assay. (C) 1994 Academic Press, Inc.