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

I. Ben-Atia, M. Fine, A. Tandler, B. Funkenstein, S. Maurice, B. Cavari, A. Gertler

Research output: Contribution to journalArticlepeer-review

33 Scopus citations

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 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 a K(d) = 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.

Original languageEnglish
Pages (from-to)155-164
Number of pages10
JournalGeneral and Comparative Endocrinology
Volume113
Issue number1
DOIs
StatePublished - 1 Jan 1999
Externally publishedYes

Keywords

  • Gilthead seabream
  • Growth hormone
  • Oral administration
  • Recombinant
  • Sparus aurata

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