title: Preparation of recombinant gilthead seabream (Sparus aurata) growth hormone and its use for stimulation of larvae growth by oral administration.
abstract: 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 in E. coli BL21 (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 beta-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 a Kd = 1.93 nM. Recombinant gsGH, like ovine placental lactogen, exhibited growth-stimulating activity when applied orally to S. aurata larvae or intraperitoneally to juvenile fish.
authors.affiliation: National Center for Mariculture, Israel Oceanographic and Limnological Research, Eilat, 88112, Israel.
authors.forename: I
authors.initials: I
authors.lastname: Ben-Atia
authors.forename: M
authors.initials: M
authors.lastname: Fine
authors.forename: A
authors.initials: A
authors.lastname: Tandler
authors.forename: B
authors.initials: B
authors.lastname: Funkenstein
authors.forename: S
authors.initials: S
authors.lastname: Maurice
authors.forename: B
authors.initials: B
authors.lastname: Cavari
authors.forename: A
authors.initials: A
authors.lastname: Gertler
mesh_terms: D000284:Administration, Oral; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006128:Growth / Q000187:drug effects*; D013006:Growth Hormone / Q000096:biosynthesis / Q000494:pharmacology*; D007457:Iodine Radioisotopes; D007814:Larva; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D010473:Perciformes / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D013006:Growth Hormone; D001618:beta-Lactamases