The prolactin of European sea bass (Dicentrarchus labrax L.): cloning of cDNA and efficient expression in Escherichia coli.
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.
Doliana
R
R
Dipartimento di Biologia, Università di Padova, Italy.
Argentini
C
C
Segat
D
D
Santarossa
P
P
Mucignat
M T
MT
Colombo
L
L
Bortolussi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001492:Bass; D015153:Blotting, Western; D003001:Cloning, Molecular; D003545:Cysteine; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011388:Prolactin / Q000096:biosynthesis* / Q000737:chemistry; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D017210:Tilapia
D017931:DNA Primers; D018076:DNA, Complementary; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D011388:Prolactin; D003545:Cysteine