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Biochem Mol Biol Int ; 33(6): 1117-26, 1994 Aug.
Article in English | MEDLINE | ID: mdl-7804137

ABSTRACT

The cDNA encoding sea bass (Dicentrarchus labrax) prolactin (sbPRL) was obtained by reverse transcription-polymerase chain reaction (RT/PCR) from pituitary RNA with degenerate primers designed on the basis of the cDNAs of the two PRLs (tPRL188 and tPRL177) from the tilapia, Oreochromis niloticus. The sbPRL cDNA encodes a preprotein of 212 amino acids composed of a putative signal peptide of 24 residues and a mature protein of 188 amino acids that is the homologue of tiPRL188. The cDNA coding for the mature protein was cloned into the pAX4a+ expression vector and expressed efficiently in Escherichia coli as a beta-galactosidase-fusion protein. To split the fusion protein, a sequence encoding the hexapeptide, (Asn-Gly)3, that contains three Asn-Gly hydroxylamine-cleavable bonds, had been previously introduced by PCR upstream of the sbPRL cDNA. N-terminal sequencing confirmed that the cleaved product corresponded to sbPRL. An antiserum raised against the recombinant hormone detected by immunoblotting a single band in sea bass pituitaries and two bands in tilapia pituitaries, suggesting the occurrence of a single PRL form in sea bass.


Subject(s)
Prolactin/biosynthesis , Recombinant Proteins/biosynthesis , Amino Acid Sequence , Animals , Base Sequence , Bass , Blotting, Western , Cloning, Molecular , Cysteine , DNA Primers , DNA, Complementary , Escherichia coli , Molecular Sequence Data , Polymerase Chain Reaction , Prolactin/chemistry , Protein Biosynthesis , Protein Sorting Signals/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Recombinant Proteins/chemistry , Sequence Homology, Amino Acid , Tilapia
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