Seiji Sugimoto

Oral administration of recombinant salmon growth hormone to rainbow trout, Oncorhynchus mykiss

Abstract Recombinant chum salmon growth hormone (rsGH) was incorporated into a polymer matrix that remains intact in acidic conditions of the stomach, but decays at higher pHs in the intestine, in an attempt to protect the hormone from proteolytic cleavage in the stomach. The GH enteric polymer matrix (GH-EPM) and free rsGH were administered to rainbow trout by admixing in food. Changes in plasma GH levels were subsequently measured by a radioimmunoassay specific for chum salmon GH. Feeding of GH-EPM in a wet food pellet resulted in significantly higher and longer elevations in plasma GH levels than that of free GH. Moreover, the GH-EPM group exhibited greater increases in length and body weight than controls. These results strongly suggest that the enteric polymer matrix protected the hormone from proteolytic enzymes in the gastrointestinal tract, allowing immunoreactive and bioactive hormone to enter the circulation and subsequently stimulate growth.

Higher culture pH is preferable for inclusion body formation of recombinant salmon growth hormone inEsherichiacoli

SummaryHigher culture pH of 7.6 was shown to be preferable for the inclusion body formation of salmon growth hormone (SGH) inEscherichiacoli harboring a recombinant plasmid. High-level formation of SGH inclusion bodies was achieved at 33°C (pH 7.6). Growth inhibition by soluble SGH was also observed.

Isolation and characterization of recombinant eel growth hormone expressed in Escherichia coli

To obtain information about the microheterogeneity of recombinant protein, recombinant eel growth hormone II (EGH) analogues expressed in Escherichia coli were isolated and characterized. The modification was classified into three types: monodeamidation of Asn, oxidation of Met and N-terminal formylation. Monodeamidated EGH was isolated by ion-exchange chromatography. The major deamidation site (Asn 147) was determined by peptide mapping using the substrate specificity of trypsin. Oxidized EGH and N-terminal-formylated EGH were isolated by reversed-phase high-performance liquid chromatography. Oxidized EGH was identified by amino acid composition analysis and N-terminal-formylated peptide by mass spectrometry.

Modulation of oligosaccharide structure of a pro-urokinase derivative (pro-UKΔGS1) by changing culture conditions of a lymphoblastoid cell line Namalwa KJM-1 adapted to serum-free medium

Pro-UKΔGS1 was designed as a long-life and thrombin-resistant derivative of pro-urokinase (pro-UK) by deleting the growth factor domain of pro-UK and introducing a glycosylation site near the thrombin cleaving site for thrombin-resistance using site-directed mutagenesis. An expression plasmid for pro-UKDGS1, pIH1UKΔGS1SEd1–5 was constructed and introduced into Namalwa KJM-1, a lymphoblastoid cell line adapted to serum-free medium, and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UKΔGS1 producer (resistant to 200 nM of MTX), clone 2–9, was selected and used for further studies.Under the conventional conditions, i.e. at 37°C in serum-free ITPSGF medium (based on RPMI-1640 medium), the oligosaccharide structure of pro-UKΔGS1 produced by clone 2–9 mainly consisted of fucose (Fuc)-containing biantennary complex-type oligosaccharide. Addition of dexamethasone (Dex), changed the carbohydrate contents in the media, and a shift down of incubation temperature caused a change in oligosaccharide structure of pro-UKΔGS1 from mainly Fuc-containing biantennary to mainly Fuc-containing tri-and tetraantennary complex-type oligosaccharide. The modulated pro-UKΔGS1 showed superiorin vivo activity for a canine femoral thrombosis formed by inserting a copper-coil.

Characterization of recombinant eel growth hormone

Abstract The characterization of purified recombinant eel growth hormone (rEGH) is described. N-Terminal sequence analysis, amino acid composition analysis, and tryptic mapping confirmed that the primary structure of rEGH was identical with that of its natural counterpart except for an additional Met at the N-terminus. Peptide mapping also revealed that rEGH had two disulphide bonds, Cys49-Cysl6O and Cysl77-Cysl85, as in mammalian growth hormones (GHs). Recombinant EGH was classified as an α-helix-rich protein, similar to mammalian GHs, from the circular dichroism spectrum. Recombinant EGH was immunochemically identical with pituitary-derived EGH. Gel filtration chromatography and light-scattering analysis indicated that rEGH exists as the monomer, as does pituitary-derived EGH.

Modulation of oligosaccharide structure of a pro-urokinase derivative (pro-UKΔGS1) by culture conditions

Pro-UKΔGS1 was designed as a stable and thrombin-resistant derivative of pro-urokinase (pro-UK) by deleting the growth factor domain of pro-UK and introducing a glycosylation site at near the thrombin cleaving site for thrombin-resistance using site-directed mutagenesis. An expression plasmid for pro-UKΔGS1, plH1UKΔGS1SEd1-5, was constructed using dhfr and pSVneo selectable markers and introduced into Namalwa KJM-1 cells. Cells resistance to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UKΔGS1 producer (resistant to 200nM MTX), clone 2–9, was selected and used for further studies.