Preparation of Recombinant Gilthead Seabream (Sparus aurata) Growth Hormone and Its Use for Stimulation of Larvae Growth by Oral Administration

• Published in: General and comparative endocrinology 1999-01, Vol.113 (1), p.155-164
• Main Authors: Ben-Atia, I, Fine, M, Tandler, A, Funkenstein, B, Maurice, S, Cavari, B, Gertler, A
• Format: Article
• Language: English
• Subjects: Administration, Oral; Animals; beta-Lactamases - metabolism; Electrophoresis, Polyacrylamide Gel; gilthead seabream; Growth - drug effects; growth hormone; Growth Hormone - biosynthesis; Growth Hormone - pharmacology; Iodine Radioisotopes; Larva; Microsomes, Liver - drug effects; Microsomes, Liver - metabolism; oral administration; Perciformes - metabolism; recombinant; Recombinant Proteins - biosynthesis; Recombinant Proteins - pharmacology; Sparus aurata
• ISSN: 0016-6480; 1095-6840
• DOI: 10.1006/gcen.1998.7192

Gilthead seabream (Sparus aurata) growth hormone (gsGH) cDNA coding for the mature protein was cloned in a pGEM-T vector and then transferred into prokaryotic expression vector pET-8 and expressed inE. coliBL21 (DE3) cells upon induction with IPTG. The expressed protein, contained within the inclusion-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 nondenaturing conditions and partial amino acid N-terminal sequence showed the purified protein to be a monomeric alanyl-gsGH. Over 90% pure bacterial β-lactamase was copurified as a by-product. Binding assays of the [125I]gsGH to gs liver microsomal fraction resulted in high specific binding characterized by aKd= 1.93 nM. Recombinant gsGH, like ovine placental lactogen, exhibited growth-stimulating activity when applied orally toS. auratalarvae or intraperitoneally to juvenile fish.