Preparation of recombinant gilthead seabream (Sparus aurata) growth hormone and its use for stimulation of larvae growth by oral administration

Gilthead seabream (Sparus aurata) growth hormone (gsGH) cDNA coding for the mature protein was cloned in a pGEM-T vector and then transferred into pro karyotic expression vector pET-8 and expressed in E. coli BL21 (DE3) cells upon induction with IPTG. The expressed protein, contained within the inclu sion-body pellet, was solubilized in 4.5 M urea, refolded at pH 11.3 in the presence of catalytic amounts of cysteine, and purified to over 98% purity , as evidenced by SDS-PAGE. Gel-filtration on a Superdex column under nonde naturing conditions and partial amino acid N-terminal sequence showed the p urified protein to be a monomeric alanyl-gsGH. Over 90% pure bacterial beta -lactamase was copurified as a by-product. Binding assays of the [I-125]g-s GH to gs liver microsomal fraction resulted in high specific binding charac terized by a K-d = 1.93 nM. Recombinant gsGH, like ovine placental lactogen , exhibited growth-stimulating activity when applied orally to S. aurata la rvae or intraperitoneally to juvenile fish. (C) 1999 Academic Press.