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 ex pressed in Escherichia coli BL21(DE3) cells upon induction with isopro pyl thiogalactoside. The expressed protein contained within the inclus ion-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 composi tion and N-terminal sequence confirmed the identity to be the predicte d protein. Binding assays of the I-125-gsIGF-I to gilthead seabream or carp (Cyprinus carpio) sera resulted in high specific binding, indica ting the existence of one or more IGF-binding proteins. In binding exp eriments to crude gilthead seabream brain homogenate, using human (h) IGF-I as a ligand, the respective IC50 value Of hIGF-I was about fourf old lower than that of gsIGF-I. Recombinant gsIGF-I exhibited mitogeni c activity in a mouse mammary gland-derived MME-L1 cell line which was similar to 200-fold lower than that of hIGF-I. 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 cel ls. In contrast, the activities of gsIGF-I and hIGF-I measured by S-35 uptake by gill arches from the goldfish (Carasrius auratus) were iden tical, indicating that the recombinant gsIGF-I is biologically active.