The prolactin of European sea bass (Dicentrarchus labrax L.): Cloning of cDNA and efficient expression in Escherichia coli

R. Doliana, C. Argentini, D. Segat, P. Santarossa, M. T. Mucignat, L. Colombo, M. Bortolussi

Research output: Contribution to journalArticle

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 β-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.

Original languageEnglish
Pages (from-to)1117-1126
Number of pages10
JournalBiochemistry and Molecular Biology International
Volume33
Issue number6
Publication statusPublished - 1994

ASJC Scopus subject areas

  • Biochemistry
  • Genetics
  • Molecular Biology

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    Doliana, R., Argentini, C., Segat, D., Santarossa, P., Mucignat, M. T., Colombo, L., & Bortolussi, M. (1994). The prolactin of European sea bass (Dicentrarchus labrax L.): Cloning of cDNA and efficient expression in Escherichia coli. Biochemistry and Molecular Biology International, 33(6), 1117-1126.