In Vivo Bioassay of Recombinant Human Growth Hormone Synthesized in B. mori Pupae (original) (raw)
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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 -lactamase was copurified as a by-product. Binding assays of the [ 125 I]g-sGH 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. 1999 Academic Press
Production and secretion of biologically active recombinant canine growth hormone by Pichia pastoris
Gene, 2004
Production of recombinant canine (Canis familiaris) growth hormone (rCFGH) by two expression systems, methanol utilization slow (Mut s) and methanol utilization plus (Mut +) based on Pichia pastoris. Led by the Saccharomyces cerevisiae a-mating type signal sequence (SS), the hormone was secreted into the culture medium in its mature and active form. The level of total proteins secreted into the medium achieved at 25 ml working volume using Erlenmeyer flasks was approximately 40 and 15 Ag/ml for Mut s and Mut + constructs, respectively. As judged by densitometry of proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the hormone produced by the fermented Mut s strain upon induction with methanol reached 24 Ag/ml, representing around 60% of the total secreted proteins and being eight times more abundant than in its Mut + counterpart. Finally, the recombinant hormone showed activity when tested in the Nb2 cell proliferation assay.
Indian Journal of Fisheries, 2015
Recombinant growth hormone of Pangasianodon hypophthalmus (rPhGH) was efficiently expressed in Escherichia coli BL 21 (DE3) cells. The expression vector pET-32a(+) was used to clone and express a 550 bp long cDNA fragment, which encodes the mature region of growth hormone. The rPhGH was expressed as a 6X HIS-tag fusion protein in E. coli upon induction by Isopropyl b-D-thiogalactoside, and formed insoluble inclusion bodies in the host cells. SDS-PAGE analysis indicated that the molecular weight of the fusion protein was about 23 kDa, which is comparable to the theoretical value of the mature growth hormone of the fish. The expressed protein was recovered by solubilising the inclusion bodies under denaturing conditions with urea and then the denatured proteins were refolded and purified on Ni-NTA column. The purified recombinant protein was confirmed by Western blot analysis using anti-His antibodies. Total yield of the refolded and purified protein was 20 mg l-1 of LB medium. Biolog...
2009
Bagi mengekspreskan gen hGH di dalam Bacillus megaterium, 3 konstrak telah direka. Konstrak-konstrak ini dinamakan sebagai Construct 1 (M7hGH), Construct 2 (R2L4sphGH) dan Construct 3 (spBsubhGH). Construct 1 direka dengan memasukkan gen pengawalatur (kodon penamat, tapak perlekatan ribosom, jujukan TAACA dan kodon pemula) yang akan diekspreskan di dalam sel. Manakala Construct 2 telah dicipta untuk diekpreskan di luar sel yang mana mengandungi isyarat peptida daripada Bacillus brevis dan gen. In order to express the hGH gene in Bacillus megaterium, 3 constructs were designed. The constructs were named as Construct 1 (M7hGH), Construct 2 (R2L4sphGH) and Construct 3 (spBsubhGH). Construct 1 was designed with regulatory features [stop codon, ribosome binding site (RBS), TAACA sequence and start codon] meant for intracellular expression. Construct 2, was created to express human growth hormone (hGH) extracellularly by using the signal peptide from Bacillus brevis
Biotechnology Letters, 2004
Giant catfish growth hormone (gcGH) cDNA was cloned and expressed in E. coli. The expected 20.5 kDa protein corresponded to the mature gcGH and was efficiently expressed. This protein was produced as inclusion bodies and comprised about 20% of total cellular proteins. The recombinant hormone promoted growth when injected intramuscularly or intraperitoneally into goldfish (Carassius auratus) at 0.1 or 1 μg soluble gcGH per g fish body wt per week. In addition, the recombinant gcGH inclusions had growth-promoting activity similar to that of the soluble form when the fish was received either by intraperitoneal injection or by oral administration.
A new human growth hormone production process using a recombinant Bacillus subtilis strain
Journal of Biotechnology, 1991
We constmcted a series of hybrid plasmids which directed the synthesis of different human growth hormone (hGH) precursor sequences in Bacillus subtilis. In addition to the 191 amino acids of the hormone, the precursors had in common an amino-termina1 extension characterized by the presence of a methionine at position 1 and of the tetrapeptide Ile-Glu-Gly-Arg preceding the first residue (Phe) of hGH.
Aquaculture, 2005
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.
Production and Bioactivity Potential of Three Recombinant Growth Hormones of Farmed Fish
Indonesian Aquaculture Journal, 2010
This study was aimed to produce recombinant growth hormone (rGH) from giant grouper (Epinephelus lanceolatus), giant gouramy (Osphronemus gouramy) and common carp (Cyprinus carpio) and compare their bioactivity potential by means of inducing the growth hormone of juvenile Nile tilapia (Oreochromis niloticus) as the model. DNA fragment encoding mature GH protein of giant grouper (El-mGH), giant gouramy (Og-mGH) and common carp (Cc-mGH) was amplified by PCR method. The purified PCR products were ligated to pCold-1 to generate pCold/El-mGH, pCold/OgmGH, and pCold/Cc-mGH protein expression vector, respectively. Each of the expression vectors was transformed into the Escherichia coli BL21. E. coli BL21 was cultured using 2xYT medium and protein production was induced by cold shock at 15±1oC for overnight. The inclusion bodies of E. coli transformants containing protein expression vector were isolated by sonication method, and rGH production was analyzed by SDS-PAGE. Juvenile of Nile tila...