Cloning and expression of somatolactin, a pituitary hormone related to growth hormone and prolactin from gilthead seabream, Sparus aurata.
A pituitary hormone, somatolactin (SL), belonging to the GH/PRL family, is produced in the intermediate lobe of the teleost pituitary. The function of this protein is uncertain. Clones coding for SL were isolated and sequenced from a gilthead seabream pituitary cDNA expression library. The nucleotide sequence of the larger cDNA isolated was 1.5 kb containing a 0.8-kb 3'-untranslated region and two potential polyadenylation signals (AATAAA). The mature polypeptide is composed of 207 amino acids, and a signal peptide of 24 residues was also found in the SL precursor. A potential N-glycosylation site Asn-Lys-Thr was identified in gilthead seabream SL. A comparison of the SL amino acid sequences of several fishes indicated that seven cysteine residues are characteristically present in all the SLs so far isolated. Six of those residues are present in homologous positions in SL and GH Sparus aurata proteins. SL and GH from S. aurata showed a 43% homology at the nucleotide level and 22% identity at the amino acid level. Expression of recombinant SL (rSL) in Escherichia coli and isolation from inclusion bodies led to a monomeric form of SL identical in electrophoretic mobility to one of the two forms of the native SL secreted from gilthead seabream pituitaries cultured in vitro. Further, a native glycosylated modified SL secreted in vitro as shown by N-glycosidase treatment was identified. Specific anti-SL antibodies that discriminate well against gilthead sea-bream GH and PRL in immunoblotting were also raised against rSL.
Astola
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Departamento de Bioquímica y Biología Molecular, Faculated de Ciencias, Universidad de Cádiz, Spain.
Pendón
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C
Ortíz
M
M
Valdivia
M M
MM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003545:Cysteine; D018076:DNA, Complementary / Q000737:chemistry; D029941:Fish Proteins; D015870:Gene Expression; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics*; D006031:Glycosylation; D008969:Molecular Sequence Data; D010473:Perciformes; D010907:Pituitary Hormones / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
D018076:DNA, Complementary; D029941:Fish Proteins; D006023:Glycoproteins; D010907:Pituitary Hormones; D011994:Recombinant Proteins; C064448:somatolactin protein, fish; D003545:Cysteine