Cloning, expression, and characterization of a recombinant gilthead seabream growth hormone.
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.
Sección Departamental de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Cádiz, Spain.
J P
JP
Martínez-Barberá
C
C
Pendón
R B
RB
Rodríguez
J
J
Pérez-Sánchez
M M
MM
Valdivia
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D013006:Growth Hormone / Q000235:genetics*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010473:Perciformes / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics
D011994:Recombinant Proteins; D013006:Growth Hormone