Browsing by Author "Funkenstein, Bruria"
Differential expression of insulin-like growth factor I and II mRNAs during embryogenesis and early larval development in rabbitfish, Siganus guttatus FG Ayson, EGT de Jesus, S Moriyama, S Hyodo, B Funkenstein, A Gertler & H Kawauchi -
General and Comparative Endocrinology, 2002 - Academic PressIn rodents, the expression of insulin-like growth factor II (IGF-II) is higher than that of insulin-like growth factor I (IGF-I) during fetal life while the reverse is true after birth. We wanted to examine whether this is also true in fish and whether IGF-I and IGF-II are differentially regulated during different stages of embryogenesis and early larval development in rabbitfish. We first cloned the cDNAs of rabbitfish IGF-I and IGF-II from the liver. Rabbitfish IGF-I has an open reading frame of 558 bp that codes for a signal peptide of 44 amino acids (aa), a mature protein of 68 aa, and a single form of E domain of 74 aa. Rabbitfish IGF-II, on the other hand, has an open reading frame of 645 bp that codes for a signal peptide of 47 aa, a mature protein of 70 aa, and an E domain of 98 aa. On the amino acid level, rabbitfish IGF-I shares 68% similarity with IGF-II. We then examined the relative expression of the two IGFs in unfertilized eggs, during different stages of embryogenesis, and in early larval stages of rabbitfish by a semiquantitative reverse transcription-polymerase chain reaction. Primers that amplify the mature peptide region of both IGFs were used and PCR for both peptides was done simultaneously, with identical PCR conditions for both. The identity of the PCR products was confirmed by direct sequencing. Contrary to published reports for seabream and rainbow trout, IGF-I mRNA was not detected in rabbitfish unfertilized eggs; it was first expressed in larvae soon after hatching. IGF-II mRNA, however, was expressed in unfertilized eggs, albeit weakly, and was already strongly expressed during the cleavage stage. mRNAs for both peptides were strongly expressed in the larvae, although IGF-II mRNA expression was higher than IGF-I expression.
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.