To investigate the N-glycosylation characteristics of recombinant human erythropoietin (rhEPO) produced by an industrial Chinese hamster ovary (CHO) cell line that is currently used in a large scale manufacturing process, we cultured this cell strain in static mode. The produced rhEPO in the culture supernatant was analyzed using isoelectric focusing (IEF) and Ricinus communis agglutinin-I (RCA-I) lectin precipitation. The lactate dehydrogenase (LDH) and sialidase activity in the serum-free supernatant were assayed as well. The analyses revealed that this cell strain could produce rhEPO with high sialic acid content, but during prolonged culture, cell viability decreased with time whilst the activity of sialidase present in the supernatant increased. The loss in rhEPO quality was due to a decrease in terminal sialic acid on the N-glycans, caused by sialidase degradation. The methods and findings in this paper serve as basis for further investigation of industrial production process.
Animals ; CHO Cells ; metabolism ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Erythropoietin ; biosynthesis ; genetics ; metabolism ; Genetic Engineering ; Humans ; N-Acetylneuraminic Acid ; metabolism ; Neuraminidase ; metabolism ; Proteolysis ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism

Cyanobacteria have become attractive hosts for renewable chemicals production. The low productivity, however, prevents it from industrial application. Reductant NAD(P)H availability is a chief hurdle for the production of reductive metabolites in microbes. To increase NADPH content in Synechocystis sp. PCC 6803, PHB synthase encoding gene phaC and phaE in Synechocystis was inactivated by replacing phaC&E genes with chloromycetin resistance cassette via homologous recombination. PCR analysis showed that mutant S.delta phaC&E with complete genome segregation was generated. The comparison between growth curves of S.wt and S.delta phaC&E indicated the knockout of phaC & phaE genes did not affect obviously the cell growth. Gas chromatography analysis showed that the accumulation of PHB in wild type was about 2.3% of the dry cell weight, whereas no PHB was detected in the mutant S.delta phaC&E. The data indicated that inactivation of PHB synthase gene phaC and phaE interrupted the synthesis of PHB. Further comparative study of wild type and mutant demonstrated that NADPH content in S.delta phaC&E was obviously increased. On the third day, the NADPH content in S.delta phaC&E was up to 1.85 fold higher than that in wild type. These results indicated that deleting PHB synthase gene phaC and phaE not only can block the synthesis of PHB, but also can save NADPH to contribute reductant sink in cyanobacteria. Hence, the engineered cyanobacterial strain S.delta phaC&E, in which carbon flux was redirected and NADPH was increased, will be a potential host strain for chemicals production in cyanobacteria.
Escherichia coli ; genetics ; metabolism ; Gene Knockout Techniques ; Hydroxybutyrates ; metabolism ; Metabolic Engineering ; Mutation ; NADP ; metabolism ; Polyesters ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Reducing Agents ; metabolism ; Synechocystis ; genetics ; metabolism

Phytochemical studies of the the herb Sarcopyramis bodinieri var. delicate (Melastomataceae) have been carried out. The compounds were separated by repeated D101 macroporous adsorption resin column combined with Sephadex LH-20, ODS, and silica gel chromatgrophy. The structures were identified on the basis of extensive spectroscopic data analysis, and by comparison of their spectral data with those reported. Eight flavonoid compounds isolated from the ethyl acetate extract was identified as isorhamnetin (1), quercetin (2), isorhamnetin-3-O-beta-D-glucopyranoside (3), quercetin-3-O-beta-D-glucopyranoside (4), isorhamnetin-3-O-(6"-acetyl)-beta-D-glucopyranoside (5), isorhamnetin-3-O-(2"-acetyl)-beta-D-glucopyranoside (6), quercetin-3-O-(6"-acetyl)-beta-D-glucopyranoside (7), and quercetin- 3-O-(6"-O-E-p-coumaroyl)-beta-D-glucopyranoside (8). All of the compounds were separated from the genus of Sarcopyramis for the first time.
Flavonoids ; analysis ; isolation & purification ; Magnetic Resonance Spectroscopy ; Melastomataceae ; chemistry ; Spectrometry, Mass, Electrospray Ionization