Expression and purification of a biologically active recombinant rabbitfish (Siganus guttatus) growth hormone
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Recombinant 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.
CitationFunkenstein, B., Dyman, D., Lapidot, Z., de Jesus-Ayson, E. G., Gertler, A., & Ayson, F. G. (2005). Expression and purification of a biologically active recombinant rabbitfish (Siganus guttatus) growth hormone.
Antibodies; Bacterial diseases; Centrifugation; Cysteine; Disease transmission; Elisa; Filtration; Fish culture; Fish diseases; Gene expression; Growth; Hormones; Husbandry diseases; Liver; Marine fish; Recombinants; Urea; pH effects; Expression vectors; Growth hormone; Inclusion bodies; Insulin-like growth factor I; Lysozyme; Escherichia coli; Siganus guttatus; Israel
We thank Dr. Sara Maurice of the Hebrew University of Jerusalem for assistance in recombinant GH purification. We also thank Fiona Pedroso, Edgel May Bayag, Hannah Cordero and the staff of the Fish Hatchery of SEAFDEC AQD for assistance in various aspects of the project. This work is supported by a grant from USAID (Grant No. TA-MOU-99-C19-001, Program in US–Israel Cooperative Development Research, Economic Growth).
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mRNA expression patterns for GH, PRL, SL, IGF-I and IGF-II during altered feeding status in rabbitfish, Siganus guttatus. Feeding time is a major synchronizer of many physiological rhythms in many organisms. Alteration in the nutritional status, specifically fasting, also affects the secretion rhythms of growth hormone (GH) and insulin-like growth factor-I (IGF-I). In this study, we investigated whether the expression patterns for the mRNAs of GH, prolactin (PRL) and somatolactin (SL) in the pituitary gland, and insulin-like growth factor I and II (IGF-I and IGF-II) in the liver of juvenile rabbitfish (Siganus guttatus) follow a rhythm according to feeding time and whether these hormone rhythms changes with starvation. Hormone mRNA levels were determined by real time PCR. The daily expression pattern for the mRNAs of GH, PRL and SL was not altered whether food was given in the morning (10:00 h) or in the afternoon (15:00 h). The daily GH mRNA expression pattern, however, was affected when food was not available for 3 days. In contrast, the daily expression pattern for IGF-I mRNA reaches its peak at roughly 5–6 h after feeding. This pattern, however, was not observed with IGF-II mRNA. During 15-day starvation, GH mRNA levels in starved fish were significantly higher than the control fish starting on the 9th day of starvation until day 15. The levels returned to normal after re-feeding. In contrast to GH, PRL mRNA levels in starved fish were significantly lower than the control group starting on the 6th day of starvation until 3 days after re-feeding. SL mRNA levels were not significantly different between the control and starved group at anytime during the experiment. Both IGF-I and IGF-II mRNA levels in starved group were significantly higher than the control fish on the 3rd and 6th day of starvation. mRNA levels of both IGF-I and II in the starved fish decreased starting on the 9th day of starvation. While IGF-I mRNA levels in the starved group continued to decrease as starvation progressed, IGF-II mRNA levels were not significantly different from the control during the rest of the starvation period. The results indicate that aside from GH and IGF-I, PRL and IGF-II are likewise involved in starvation in rabbitfish.
Milkfish (Chanos chanos) growth hormone cDNA cloning and mRNA expression in embryos and early larval stages. EGT de Jesus, FG Ayson, Y Amemiya, S Moriyama, S Hyodo, T Hirano & H Kawauchi -
Aquaculture, 2002 - ElsevierIn an attempt to understand growth regulation in milkfish, the milkfish growth hormone (GH) and its cDNA were characterized and the expression of GH mRNA in embryos and larvae was examined by RT-PCR. The milkfish GH was purified from an alkaline extract of the pituitary by reverse-phase high-performance liquid chromatography and detected as an immuno-positive protein with anti-salmon GH serum. The complete sequence of milkfish pre-GH was determined by cDNA cloning and nucleotide sequencing. On the basis of the N-terminal amino acid analysis of the native protein, the pre-GH was found to consist of a signal peptide of 22 amino acids and a mature protein of 188 amino acids. Milkfish GH shows higher amino acid sequence identity with GHs of carps (91–94%) and salmonids (70%) than with GHs of more advanced teleosts (<60%) in good accordance with its taxonomic position in teleosts. It has five half Cys residues, four of which are at positions homologous with those of other known GHs and the extra Cys with those of carp GHs. The molecular weight of milkfish GH was estimated to be 22 kDa, which is comparable to the theoretical value. This suggests that milkfish GH is a simple protein, although it has two potential N-glycosylation sites. Semiquantitative RT-PCR showed that GH mRNA expression was relatively weak in embryos and newly hatched larvae but was already strong in 2-day old and older larvae.
Conference paperAC Emata - In TU Bagarinao & EEC Flores (Eds.), Towards sustainable aquaculture in Southeast Asia and Japan: Proceedings of the Seminar-Workshop on Aquaculture Development in Southeast Asia, Iloilo City, Philippines, 26-28 July, 1994, 1995 - Aquaculture Department, Southeast Asian Fisheries Development CenterMost of the fish research at SEAFDEC AQD in 1992-1994 was on milkfish. Studies were conducted on year-round spawning through hormonal or environmental manipulation; optimum lipid and protein levels and ration size for captive broodstock; and the influence of spawner age on reproductive performance. The economics of hatchery operations, alone or integrated with broodstock as a commercial enterprise, was assessed. Mass production of larvae was refined with the use of commercial or SEAFDEC-formulated larval diets. Alternative rearing schemes in large tanks and ponds were tried. Hatcheryproduced and wild-caught larvae were compared in terms of growth and production in experimental nursery and grow-out ponds. Supplemental diets for brackishwater grow-out culture were formulated. Studies on broodstock management of grouper Epinephelus spp. included lipid enrichment of the diet and hormonal induction of sex inversion. Seed production techniques were developed but survival rates were low. Grouper culture was found economically feasible in experimental ponds with 'trash' fish as feed. The mangrove red snapper Lutjanus argentimaculatus was successfully induced to spawn with injection of human chorionic gonadotropin. Initial larval rearing trials were successful but survival rates must be improved. Hormonal manipulation of spawning of the Asian sea bass Lates calcarifer allows seed production during most of the year. Photoperiod manipulation leads to maturation of females, but not males, beyond the natural breeding season (April-November). Nursery rearing of 9 mm juveniles is feasible in floating net cages with night lights that attract food zooplankton. The requirements of sea bass for lipid, protein, carbohydrates, and essential amino acids were determined. In the rabbitfish Siganus guttatus, weekly injections of luteinizing hormone releasing hormone analogue (LHRHa) sustains milt production for three weeks. Thyroid hormones injected into broodstocks improved the growth of larvae to day 7. Induced spawning techniques for the Asian catfish Clarias macrocephalus were refined by determining the seasonal responsiveness to LHRHa and pimozide injections and testing for pheromonal induction of spontaneous spawning. The optimum insemination rate was determined and egg hatchability was enhanced by removal of the adhesive coat before incubation. Several practical diets for catfish during grow-out culture were tested against 'trash' fish. The broodstock management for bighead carp Aristichthys nobilis was studied. Cage-reared juveniles from cage-reared broodstock showed the best growth. To improve the reproductive performance, the broodstock diets were supplemented with vitamins A, C, and E. Research on tilapias focused on genetics and strain selection. Several strain testing procedures for Nile tilapia were evaluated in their efficiency to detect economically important strain differences. Reference lines were developed from two existing red tilapia strains to measure and reduce the effects of uncontrolled nongenetic variables in strain evaluation experiments with Nile tilapia. The tolerance of two Nile tilapia strains to heavy metals was similar when gauged by the 24-hour and 96-hour lethal concentration and by fish growth, survival, and reproductive performance. In a separate study, four strains of red tilapia showed generally higher seed production when reared in tanks than in cages. Improvements in the feed and feeding management for Nile tilapia were also studied. Intensive tilapia farming and feeding have led to oxygen depletion and fish kills in Sampaloc Lake. To rehabilitate the lake, it is imperative to reduce the farming area from 30 to 6 hectares; stop the use of commercial feeds; and remove the water hyacinths and other debris. Fish kills in Laguna de Bay have also become serious in recent years, and a review of the occurrences, losses, and possible causes is currently being conducted. Studies on the epizootic ulcerative syndrome of snakeheads in Laguna de Bay have yet to pinpoint the pathogen. Skin lesions in tilapias in several ponds and lakes in the country were found to be due to bacteria.