Recombinant gilthead seabream (Sparus aurata) insulin-like growth factor-I: subcloning, expression in Escherichia coli, purification and characterization.

Gilthead seabream (Sparus aurata) insulin-like growth factor-I (gsIGF-I) cDNA coding for the mature protein was cloned in a pGEM-3Z vector, and then transferred into prokaryotic expression vector pET-11a and expressed in Escherichia coli BL21(DE3) cells upon induction with isopropyl thiogalactoside. The expressed protein contained within the inclusion-body pellet was solubilized in 4.5 M urea, refolded for 24 h at pH 11.3 in the presence of catalytic amounts of cysteine and purified to over 98% purity, as a monomeric methionyl-gsIGF-I. Amino acid composition and N-terminal sequence confirmed the identity to be the predicted protein. Binding assays of the 125I-gsIGF-I to gilthead seabream or carp (Cyprinus carpio) sera resulted in high specific binding, indicating the existence of one or more IGF-binding proteins. In binding experiments to crude gilthead seabream brain homogenate, using human (h) IGF-I as a ligand, the respective IC50 value of hIGF-I was about fourfold lower than that of gsIGF-I. Recombinant gsIGF-I exhibited mitogenic activity in a mouse mammary gland-derived MME-L1 cell line which was approximately 200-fold lower than that of hIGF-1. Binding experiments to intact MME-L1 cells suggests that this difference most likely results from a correspondingly lower affinity for IGF-I receptor in these cells. In contrast, the activities of gsIGF-I and hIGF-I measured by 35S uptake by gill arches from the goldfish (Carassius auratus) were identical, indicating that the recombinant gsIGF-I is biologically active.

Nucleotide sequence of the promoter region of Sparus aurata insulin-like growth factor I gene and expression of IGF-I in eggs and embryos.

A genomic fragment of 3.1 kb, containing the promoter region of Sparus aurata insulin-like growth factor I (IGF-I) gene has been cloned and sequenced. This fragment contains exon 1 and exon 2 of Sparus aurata IGF-I gene. These two exons are identical in organization to the reported chum salmon IGF-I gene. Exon 1 contains 5' untranslated region and part of the signal peptide. Exon 2 codes for part of the signal peptide and part of the B domain. Nested polymerase chain reaction (PCR) revealed that a fragment was amplified from liver RNA when a common primer in the translated region and a primer located between 375 and 395 nucleotides upstream of the first methionine were used. No such amplification was obtained when the primer was located between 414 and 434 nucleotides upstream of the first methionine, suggesting that the first exon in Sparus IGF-I gene starts 400-410 nucleotides upstream of the first methionine. Expression of IGF-I mRNA was studied in Sparus aurata using reverse transcription (RT)-PCR. An amplified fragment was found in unfertilized eggs and in embryos 4, 8, and 12 hours after fertilization, when oligonucleotides specific for Sparus aurata IGF-I cDNA were used. A similar fragment was found when adult liver or one-day larval RNA were used. This fragment hybridized in a Southern blot to salmon IGF-I cDNA. These results demonstrate that the structure of IGF-I gene has been conserved in teleosts and IGF-I transcripts are present in fish during embryogenesis, probably of maternal origin.