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 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.
Ben-Atia
I
I
National Center for Mariculture, Israel Oceanographic and Limnological Research, Eilat, 88112, Israel.
Fine
M
M
Tandler
A
A
Funkenstein
B
B
Maurice
S
S
Cavari
B
B
Gertler
A
A
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
D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D013006:Growth Hormone; D001618:beta-Lactamases