Doliana R, Argentini C, Segat D, Santarossa P, Mucignat M T, Colombo L, Bortolussi M
Dipartimento di Biologia, Università di Padova, Italy.
Biochem Mol Biol Int. 1994 Aug;33(6):1117-26.
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.
