Browsing by Author "Dyman, Dyman"
Expression and purification of a biologically active recombinant rabbitfish (Siganus guttatus) growth hormone B Funkenstein, D Dyman, Z Lapidot, EG de Jesus-Ayson, A Gertler & FG Ayson -
Aquaculture, 2005 - ElsevierRecombinant rabbitfish growth hormone (rfGH) protein was expressed in Escherichia coli, BL21(DE3) cells. The cDNA encoding the mature protein of rfGH was first cloned in pGEM-Teasy vector and then transferred to pET-3d expression vector. Expression in E. coli cells was then induced by IPTG (0.4 mM). Inclusion bodies (IB) containing the expressed protein were purified by treating bacterial cells pellet with lysozyme followed by repeated washings in cold water containing Triton X-100, sonication, and centrifugation. IB were then solubilized in 4.5 M urea, refolded at pH 11.3 in the presence of catalytic amounts of cysteine and purified by Q-Sepharose column. Gel filtration on Superdex column showed the purified protein to be a monomeric GH. Based on SDS–PAGE, the purity of the recombinant rfGH preparation is approximately 98%. The recombinant rfGH was tested for its biological activity both in vitro, by its ability to stimulate IGF-I mRNA expression in the liver, and in vivo, by its ability to accelerate growth in rabbitfish fry injected with the hormone. A significant increase in growth was observed in rabbitfish fry given the recombinant hormone. Polyclonal antibody raised against the native rfGH immunoreacted with the recombinant rfGH in Western blots and in ELISA, indicating the suitability of these reagents for future quantification of GH in rabbitfish plasma.