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161.
Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells 总被引:6,自引:0,他引:6
Alain Garnier Johanne Côté Isabelle Nadeau Amine Kamen Bernard Massie 《Cytotechnology》1994,15(1-3):145-155
Human 293S cells, a cell line adapted to suspension culture, were grown to 5×106 cells/mL in batch with calcium-free DMEM. These cells, infected with new constructions of adenovirus vectors, yielded as much as 10 to 20% recombinant protein with respect to the total cellular protein content. Until recently, high specific productivity of recombinant protein was limited to low cell density infected cultures of no more than 5×105 cells/mL. In this paper, we show with a model protein, Protein Tyrosine Phosphatase 1C how high product yield can be maintained at high cell densities of 2×106 cells/mL by a medium replacement strategy. This allows the production of as much as 90 mg/L of active recombinant protein per culture volume. Analysis of key limiting/inhibiting medium components showed that glucose addition along with pH control can yield the same productivity as a medium replacement strategy at high cell density in calcium-free DMEM. Finally, the above results were reproduced in 3L bioreactor suspension culture thereby establishing the scalability of this expression system. The process we developed is used routinely with the same success for the production of various recombinant proteins and viruses.Abbreviations CFDMEM
calcium-free DMEM
- CS
bovine calf serum
- hpi
hours post-infection
- J+
enriched Joklik medium
- MLP
major late promoter
- MOI
multiplicity of infection (# of infectious viral particle/cell)
- q
specific consumption rate (mole/cell.h)
- pfu
plaque forming unit (# of infectious viral particle)
- Y
yield (g/E6 cells or mole/cell) 相似文献
162.
Vascular smooth muscle cell membranes from prehypertensive rats of the Milan hypertensive strain (MHS) were used to examine adenylyl cyclase activity and its regulation by guanine nucleotide regulatory proteins (G-proteins). Basal adenylyl cyclase activity was similar in MHS and Milan normontensive strain (MNS) membranes. Forsokolin (10?4 M) produced a significantly greater stimulatory response in MHS membranes, but this was not observed with NaF (10?2 M). Isoporterenol (10?4 M) caused a significantly decreased stimulation of adenylyl cyclase activity in MHS membranes, while prostaglandin E1 (10?5 M) produced similar responses in the two strains. Gi function and GTP responses, as observed by biphasic effects of GTP on isoproterenol-stimulated membranes, were similar in both strains. The levels of Gi2α and Gqα/G11α were similar in the two strains, while the levels of Gsα (44 and 42 kDa forms) and the β-subunit were significantly reduced by ~20% in MHS membranes. The α-subunit of Gi3 was dramatically reduced by ~80% in MHS membranes. The affinities of β-adrenergic receptors for the antagonist, cyanophindolol, were similar in the two strains; however, the number of β-adrenoceptors was substantially reduced in MHS membranes. These findings may be of relevance to altered vascular reactivity and transmembrane ion distribution observed in the MHS. 相似文献
163.
Effects of fire on water and salinity relations of riparian woody taxa 总被引:12,自引:0,他引:12
Water and salinity relations were evaluated in recovering burned individuals of the dominant woody taxa from low-elevation riparian plant communities of the southwestern U.S. Soil elemental analyses indicated that concentrations of most nutrients increased following fire, contributing to a potential nutrient abundance but also elevated alluvium salinity. Boron, to which naturalized Tamarix ramosissima is tolerant, was also elevated in soils following fire. Lower moisture in the upper 30 cm of burned site soil profiles was attributed to shifts in evapotranspiration following fire. Higher leaf stomatal conductance occurred in all taxa on burned sites. This is apparently due to higher photosynthetic photon flux density at the midcanopy level and may be partially mitigated by reduced unit growth in resprouting burned individuals. Predawn water potentials varied little among sites, as was expected for plants exhibiting largely phreatophytic water uptake. Midday water potentials in recovering Salix gooddingii growing in the Colorado River floodplain reached levels which are considered stressful. Decreased hydraulic efficiency was also indicated for this species by examining transpiration-water potential regressions. Recovering, burned Tamarix and Tessaria sericea had enriched leaf tissue 13C relative to unburned controls. Higher water use efficiency following fire in these taxa may be attributed to halophytic adaptations, and to elevated foliar nitrogen in Tessaria. Consequently, mechanisms are proposed which would facilitate increased community dominance of Tamarix and Tessaria in association with fire. The theory that whole ecosystem processes are altered by invading species may thus be extended to include those processes related to disturbance. 相似文献
164.
Active transport systems in bacteria can be divided into two groups: those that are osmotic shock-resistant with one single membrane protein, and those that are shock-sensitive and have a membrane-bound protein complex plus a soluble periplasmic protein. Whether the bacterial assimilatory nitrate transport falls into the one or the other of these two groups has not been studied before. We report that nitrate uptake by the strictly aerobic, N2 -fixing heterotrophic bacterium Azotobacter chroococcum is sensitive to osmotic shock. The polypeptide composition of cytoplasmic membranes changes in response to the nitrogen source available to the cells. Incorporation of [35 S]-methionine into proteins as well as use of the A. chroococcum TRI mutant, which is defective in nitrate transport, and the A. choococcum MCD1 strain, a mutant unable to use nitrate as a nitrogen source, suggest that nitrate transport into A. chroococcum cells is mediated by a multicomponent system tightly bound to the cytoplasmic membrane. 相似文献
165.
Three protocols for the determination of inorganic and organic sulfur fractions were tested for their suitability to estimate total indigenous organic sulfur (Sorg) and35Sorg formed from added35SO4
2– in sediments of chemically dilute lakes in the ELA. The protocols tested have all been reported in the literature. It was found that two protocols involving sequential analyses for S fractions following acid treatment gave estimates of both Sorg and35Sorg up to 87% lower than a non-sequential protocol. The low estimates were largely due to hydrolysis and solubilization of solid phase S which was then removed in a rinsing step. The non-sequential protocol, in which total reduced inorganic sulfur and total sulfur were determined on separate aliquots, is recommended as the most reliable of the three. Individual analyses in this protocol were verified for these lake sediments using a variety of S standards. 相似文献
166.
Emile CL. Marnette Harm Houweling Herman Van Dam Jan Willem Erisman 《Biogeochemistry》1993,23(2):119-144
The chemical composition of surface waters of two Dutch moorland pools and of incident precipitation, was monitored from 1982
to 1990. For this period, sulfur and water budgets were calculated using a hydrochemical model developed for well-mixed non-stratifying
lakes. Total atmospheric deposition of S decreased significantly after 1986 at both locations. A model describing the sulfur
budget in terms of input, output and reduction/oxidation processes predicted a fast decrease of pool water SO4
2− concentrations after a decrease of atmospheric input. However, SO4
2− concentrations in the surface water was lowered only slightly or remained constant. Apparently a source within the lake caused
the unexpectedly high SO4
2− concentrations. The possible supply of SO4
2− from the sediment through regulation by (K-)Al-SO4 containing minerals or desorption of SO4
2− from positively charged surfaces in the sediment was evaluated. Solubility calculations of pore water with respect to alunite,
basaluminite and jurbanite indicated that SO4
2− concentration was not regulated by these minerals. It is suggested here (1) that desorption of SO4
2− from peaty sediments may account for the estimated SO4
2− supply provided that the adsorption complex is periodically recharged by partial oxidation of the upper bottom sediments
and (2) that because of exposure of a part of the pool bottom to the atmosphere during dry summers and subsequent oxidation
of reduced S, the amount of SO4
2− may be provided which complements the decreasing depositional SO4
2− input. In future research these two mechanisms need to be investigated. 相似文献
167.
M. C. Daunay M. H. Chaput D. Sihachakr M. Allot F. Vedel G. Ducreux 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1993,85(6-7):841-850
Summary In order to produce fertile somatic hybrids, mesophyll protoplasts from eggplant were electrofused with those from one of its close related species, Solanum aethiopicum L. Aculeatum group. On the basis of differences in the cultural behavior of the parental and hybrid protoplasts, 35 somatic hybrid plants were recovered from 85 selected calli. When taken to maturity either in the greenhouse or in the field, the hybrid plants were vigorous, all rapidly overtopping parental individuals. The putative hybrids were intermediate with respect to morphological traits, and all of their organs were larger, particularly the leaves and stems. DNA analysis of the hybrids using flow cytometry in combination with cytological analysis showed that 32 were tetraploids, 1 hexaploid and 2 mixoploids. The hybrid nature of the 35 selected plants was confirmed by a comparison of the isoenzyme patterns of isocitrate dehydrogenase (Idh), 6-phosphogluconate dehydrogenase (6-Pgd) and phosphoglucomutase (Pgm). Chloroplast DNA (ctDNA) restriction analysis using Bam HI revealed that among the 27 hybrid plants analyzed, 10 had S. aethiopicum patterns and the 17 remaining hybrids exhibited bands identical with those of eggplant without any changes. All of the somatic hybrid plants flowered. Both parental plants had 94% stainable pollen, while the hybrids varied widely in pollen viability ranging from 30% to 85%. The somatic hybrids showed high significant variation in fruit production. Nevertheless, there was a tendency for low fertility to be associated often with S. aethiopicum chloroplast type and/or with an abnormal ploidy level, while good fertility was mostly associated with the tetraploid level and eggplant chloroplasts. Interestingly, 2 tetraploid somatic hybrid clones were among the most productive, yielding up to 9 kg/plant. As far as the fertility of the F1 sexual counterpart was concerned, only 2 fruits of 50 g were obtained. Hybrid fertility in relation to phylogenetic affinities of the fusion partners is discussed. 相似文献
168.
169.
170.
For phylogenetic analysis of the higher fungi, we sequenced the nuclear small subunit rRNA (18S rRNA) gene fromTaphrina populina, the type species of the genusTaphrina, andProtomyces lactucae-debilis. The molecular phylogeny inferred from these 2 sequences and 75 sequences from the DNA data bank divided the Ascomycota into three major lineages: the hemiascomycetes, the euascomycetes, and the archiascomycetes, newly described herein. The former two lineages are monophyletic, whereas the archiascomycetes, which originated first and are comprised ofTaphrina, Protomyces, Saitoella, Schizosaccharomyces, andPneumocystis, may not be monophyletic. Among the archiascomycetes, theTaphrina/Protomyces branch is monophyletic. Confirmation of the archiascomycetes as a monophyletic taxonomic class will require comparison of additional genetically defined characters.This work was supported in part by grants 05454030 from the Ministry of Education, Science, and Culture of Japan (to J. S.) and 4369 from the Japan Society for the Promotion of Science Fellowship Programs (to H. N.). 相似文献