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1.
Effects of medium quality on the expression of human interleukin-2 at high cell density in fermentor cultures of Escherichia coli K-12 总被引:2,自引:0,他引:2
We examined the ability of transformed Escherichia coli cells in fermentor cultures to accumulate interleukin-2 (IL-2) intracellularly under temperature-regulated control of the phage lambda pL promoter. Induction of expression was undertaken at different culture optical densities, and specific IL-2 accumulation was found to decrease with increasing cell density at induction. Induction at higher culture optical densities was also accompanied by decreased growth during induction and increased acetate accumulation in the culture medium. Experiments were undertaken to study the effect of replacing spent medium by perfusion with fresh medium both before induction and during IL-2 expression at high cell density. Improved IL-2 expression was seen only when perfusion was continued past 1.6 h after the start of induction, and it was accompanied by a significant reduction in acetate buildup. Further improvements were not seen when perfusion was continued beyond hour 3 of induction. Replenishing medium components and decreasing the concentration of diffusible inhibitors before induction did not alleviate acetate buildup, growth limitation, or limitation of IL-2 synthesis. These results suggested that accumulation of diffusible inhibitors such as acetate during induction may be a significant factor limiting IL-2 expression in high-density cultures, but other factors intrinsic to the organism or the protein also played a major role. 相似文献
2.
Improved expression of human interleukin-2 in high-cell-density fermentor cultures of Escherichia coli K-12 by a phosphotransacetylase mutant. 总被引:3,自引:0,他引:3
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K A Bauer A Ben-Bassat M Dawson V T de la Puente J O Neway 《Applied microbiology》1990,56(5):1296-1302
A fluoroacetate-resistant mutant of Escherichia coli K-12 (MM-294) accumulated less acetate in the medium during growth to high cell density in fermentor cultures and was shown to be defective in its phosphotransacetylase activity. The mutant had an improved ability to continue growing during induction of interleukin-2 (IL-2) synthesis, and in fermentor cultures it gave a higher level of specific IL-2 accumulation than its parent during expression under control of the temperature-sensitive pL promoter. In flask cultures at lower cell density, the mutant again produced less acetate than the parent, although both showed a much lower level of acetate accumulation than that seen in fermentors at high cell density. Both showed a higher specific expression level of IL-2 in flask cultures, and there was a greater difference between the mutant and its parent in the final extent of specific IL-2 accumulation in fermentor cultures compared with flask cultures. Thus, the concentration of acetate in the medium, which was much higher in fermentor cultures (greater than or equal to 300 mM after 5 h of induction) than in flask cultures (less than or equal to mM) of the parent organism, was a significant factor in limiting expression of the heterologous protein product, IL-2. The acetate kinase-phosphotransacetylase pathway was therefore a major source of acetate formation in these cultures. Blocking this pathway improved accumulation of IL-2 and did not slow growth. 相似文献
3.
K A Bauer A Ben-Bassat M Dawson V T de la Puente J O Neway 《Applied and environmental microbiology》1990,56(5):1296-1302
A fluoroacetate-resistant mutant of Escherichia coli K-12 (MM-294) accumulated less acetate in the medium during growth to high cell density in fermentor cultures and was shown to be defective in its phosphotransacetylase activity. The mutant had an improved ability to continue growing during induction of interleukin-2 (IL-2) synthesis, and in fermentor cultures it gave a higher level of specific IL-2 accumulation than its parent during expression under control of the temperature-sensitive pL promoter. In flask cultures at lower cell density, the mutant again produced less acetate than the parent, although both showed a much lower level of acetate accumulation than that seen in fermentors at high cell density. Both showed a higher specific expression level of IL-2 in flask cultures, and there was a greater difference between the mutant and its parent in the final extent of specific IL-2 accumulation in fermentor cultures compared with flask cultures. Thus, the concentration of acetate in the medium, which was much higher in fermentor cultures (greater than or equal to 300 mM after 5 h of induction) than in flask cultures (less than or equal to mM) of the parent organism, was a significant factor in limiting expression of the heterologous protein product, IL-2. The acetate kinase-phosphotransacetylase pathway was therefore a major source of acetate formation in these cultures. Blocking this pathway improved accumulation of IL-2 and did not slow growth. 相似文献
4.
Sp2/0 hybridoma cells die principally by apoptosis in batch culture. We have found that cultures of the Sp2/0 hybridoma exhibit increased viability in response to interleukin 6 (IL-6) supplementation relative to control cultures during serum shiftdown experiments. When shifted from a medium containing 10% fetal bovine serum (FBS) to a medium with 1% FBS, IL-6 supplemented cultures displayed viabilities and viable cell densities similar to control cultures containing 10% FBS. The degree of the survival response induced varied in accordance with the severity of the shiftdown, as cells resuspended in a high serum medium showed little observable enhancement in viability. The extension in culture viability was not accompanied by an observable decrease in growth relative to control cultures, indicating that the effect was not a consequence of growth inhibition. These results suggest the existence of serum components with behavior functionally similar to IL-6, with respect to enhancing cell survival, and that under certain experimental conditions IL-6 serves as a survival factor. In contrast to the extended viability displayed by cultures supplemented with IL-6, Sp2/0 cultures transfected with IL-6 cDNA expression vectors displayed a growth inhibitory response relative to control cultures. This inhibitory response was characterized by an extended lag phase following inoculation, and a decrease in batch culture cell yield. The depression in cell yield varied with serum concentration, with the largest depression occurring at high serum concentrations. We conclude that interactions between components in serum, presumably growth factors, and cytokines play an important role in altering the behavior of industrially relevant cell lines in culture. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 439-446, 1997. 相似文献
5.
The most common strategy to produce recombinant proteins using Escherichia coli as expression vector is fed-batch culture, since high cell density cultures strategies have successfully been applied. Several methodologies to limit the specific growth rate in order to control E. coli metabolism have been defined, demonstrating that cultures can be grown under glucose limitation up to high cell densities without accumulation of acetic acid. However, under induction conditions it has been observed that E. coli metabolism is reorganized again and leads to acetic acid accumulation, causing inhibition of cell growth and decreasing protein expression efficiency.We propose a double limitation strategy (glucose and IPTG) for E. coli fed-batch cultures to avoid the deregulation of the metabolism in the induction phase. Reducing the concentration of IPTG while keeping glucose growth limitation, the accumulation of acetic acid decreased. At an IPTG concentration of 0.03 mmol/g DCW no accumulation of acetic acid was observed during the induction phase, in contraposition to what has normally been observed.Although a slight reduction of protein expression rate was observed when applying this double limitation strategy, the bioprocess volumetric productivity was enhanced due to the capability to prolong the induction phase, reaching higher levels of protein production. Another advantage of this strategy is the reduction of media cost due to the lower level of IPTG used. 相似文献
6.
Between feast and famine: endogenous inducer synthesis in the adaptation of Escherichia coli to growth with limiting carbohydrates. 总被引:2,自引:1,他引:1
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Escherichia coli adapted to growth with low carbohydrate concentrations bypassed the requirement for exogenous inducer with at least three well-studied sugar regulons. Induction of mgl and gal genes became independent of added galactose in bacteria approaching stationary phase or during continuous culture with micromolar glucose in the medium. Bacteria became independent of exogenous induction because endogenous galactose and cyclic AMP (cAMP) pools were sufficient for high expression of mgl and gal genes under glucose limitation. Limitation-stimulated induction of mgl was dependent on a functional galETK operon for synthesis of the inducer galactose. Intracellular galactose levels were maximal not during starvation (or slow steady-state growth rates approaching starvation) but at fast growth rates with micromolar glucose. The extent of mgl/gal induction correlated better with inducer availability than with cAMP concentrations under all conditions tested. Endogenous inducer accumulation represents an adaptation to low-nutrient environments, leading to derepression of high-affinity transport systems like Mgl essential for bacterial competitiveness at low nutrient concentrations. 相似文献
7.
Pseudomonas oleovorans (ATCC 29347) was grown in batch and chemostat cultures with citrate, hexanoate, heptanoate, octanoate, and nonanoate as single carbon substrates. The growth medium for batch cultures was adjusted such that nitrogen (NH(4)(+)) limitation terminated the exponential-growth phase. During batch cultivation with octanoate or nonanoate the biomass continued to increase after depletion of ammonium due to the accumulation of medium-chain-length poly[(R)-3-hydroxyalkanoates] (mcl-PHAs). Additionally, a significant rate of mcl-PHA accumulation was also observed in the exponential-growth phase of batch cultures. It is well known that the accumulation of reserve materials is strongly dependent on the ratio of nutrients (here of carbon, C, and of nitrogen, N) and that in a batch culture the ratio of C:N is continuously changing. Therefore, we have also investigated the effect of defined ratios of C:N under constant cultivation conditions, namely at a fixed dilution rate (D) in a chemostat fed with different medium C:N ratios. These experiments were performed at a constant D of 0.2 h(-1). The concentration of the nitrogen source in the inflowing medium (N()) was kept constant, while its carbon concentration (C()) was increased stepwise, resulting in an increase of the medium carbon to nitrogen ratio (C()/N() ratio). The culture parameters and the cell composition of steady-state cultures were determined as a function of the C()/N() ratio in the feed medium. Mcl-PHA accumulation was detected during growth with the fatty acids, and three distinct regimes of growth limitation were discovered: In addition to carbon limitation at low, and nitrogen limitation at high C()/N() ratios, an intermediate growth regime of simultaneous limitation by carbon and nitrogen was detected where both substrates were used to completion. The width of this dual-nutrient-limited growth regime was dependent on the change in the yield factors for carbon and nitrogen (Y(X/C), Y(X/N)) measured during single-nutrient-limited growth. 相似文献
8.
研究了不同碳源(葡萄糖、乳酸和乙酸)以及IPTG诱导对工程菌MM2表达霍乱毒素B亚单位(CTB)的影响,ctb基因位于lac启动子的下游。在YC培养基中分别加入0.048mol/L的葡萄糖、0.102mol/L的乳酸和0.167mol/L的乙酸,它们在完全氧化后可产生相同的能量。结果表明,加入葡萄糖会大幅度降低ctb基因的表达水平,其原因和培养过程中pH值下降有关;加入乳酸可提高ctb基因表达水平1.15倍,且不抑制菌体生长;加入乙酸可提高ctb基因的表达水平0.9倍,但对菌体生长有抑制作用。不同时间及不同浓度的IPTG诱导未能提高ctb基因的表达水平,说明lac启动子对ctb基因的表达没有影响或影响很小。 相似文献
9.
Heterologous endo-beta-1,4-xylanase was produced by Pichia stipitis under control of the hypoxia-inducible PsADH2-promoter in a high-cell-density culture. After promoter induction by a shift to oxygen limitation, different aeration rates (oxygen transfer rates) were applied while maintaining oxygen-limitation. Initially, enzyme production was higher in oxygen-limited cultures with high rates of oxygen transfer, although the maximum xylanase activity was not significantly influenced. Amino acid supplementation increased the production of the heterologous endo-beta-1,4-xylanase significantly in highly aerated oxygen-limited cultures, until glucose was depleted. A slight second induction of the promoter was observed in all cultures after the glucose had been consumed. The second induction was most obvious in amino acid-supplemented cultures with higher oxygen transfer rates during oxygen limitation. When such oxygen-limited cultures were shifted back to fully aerobic conditions, a significant re-induction of heterologous endo-beta-1,4-xylanase production was observed. Re-induction was accompanied by ethanol consumption. A similar protein production pattern was observed when cultures were first grown on ethanol as sole carbon source and subsequently glucose and oxygen limitation were applied. Thus, we present the first expression system in yeast with a sequential double-inducible promoter. 相似文献
10.
The presence of serum in cell culture raises safety problems for the production of biologicals, thus a new serum-free medium (MDSS2) was developed. The evaluation of this medium for the growth of different cell lines (BHK-21 C13, BSR and Vero) has shown that cells grew in this medium similarly to standard serum-containing medium, independently of the culture system used: in static (as monolayer) as well as in agitated systems (in suspension in spinner and perfusion reactors). BHK-21 and BSR cells grew as aggregate cultures and could proliferate in both static and agitated culture systems. Vero cells stayed attached to a substrate and proliferated equally in static and in agitated microcarrier-culture systems. The cell densities obtained with BHK-21 cells depended only on the culture system used. They ranged from 2–3×106 to 6–12×106 cells per ml for static batch and perfusion reactor cultures respectively. The cell concentration was 3 to 6 times higher than in classical cultures performed in serum-containing medium. The cell densities obtained with Vero cells were indistinguishable from those obtained in serum-containing medium, whatever the cell culture system used. These cell lines have been used for the production of rabies virus. With respect to BHK-21 and BSR, similar production rates of rabies glycoprotein have been found as in the standard roller bottle process. The production of rabies virus and of viral glycoprotein by Vero cells cultivated in serum-free medium was augmented 1.5-fold and 2.5-fold, respectively, when compared to serum-containing medium.A recombinant BHK-21 cell line, producing human IL-2, can also proliferate in MDSS2, after addition of insulin. The specific IL-2 production rate was augmented 3–4 fold in comparison to serum-containing medium.For the cells tested, the MDSS2 serum-free medium is a good growth and production medium. Its use for cultivating other cell lines and/or for the production of other biologicals is discussed. 相似文献
11.
A perfusion culture system was developed to investigate the oxygenation of high-density hybridoma cell cultures. The culture system was composed of a stirred-tank bioreactor and an external microfiltration hollow fiber cartridge for medium perfusion. Cell growth and antibody production were examined with large bubble ( approximately 5 mm in diameter), micron-sized bubble ( approximately 80 mum in diameter), and silicone tubing oxygenation techniques. Comparable cell growth and monoclonal antibody (MAb) production were found for both the micron-sized and large oxygenation methods, provided that large bubbles were enriched with pure oxygen. Relatively low cell growth and MAb production were attained with the bubble-free silicone tubing oxygenation. It is concluded that direct bubble oxygenation can be applied successfully in high-density animal cell cultures, provided that the culture medium is supplemented with Pluronic F-68. The accumulation of ammonia in the culture medium rather than oxygen limitation was found to be one of the possible problems that eventually inhibited cell growth. This and the fouling of the filtration cartridge during long-term cultivation were found to be more problematic than simple bubble oxygenation of high-density cell culture. The micron-sized bubble oxygenation method is highly recommended for high-density animal cell cultures, provided that Pluronic F-68 is supplemented into the culture medium. (c) 1993 John Wiley & Sons, Inc. 相似文献
12.
Microcin J25 is a 2,107-Da, plasmid-encoded, cyclopeptide antibiotic produced by Escherichia coli. We have isolated lacZ fusions to mcjA (encoding the 58-amino-acid microcin precursor) and mcjB and mcjC (which are required for microcin maturation), and the regulation of these fusions was used to identify factors that control the expression of these genes. The mcjA gene was found to be dramatically induced as cells entered the stationary phase. Expression of mcjA could be induced by resuspending uninduced exponential-phase cells in spent supernatant obtained from an early-stationary-phase culture. Induction of mcjA expression was not dependent on high cell density, pH changes, anaerobiosis, or the buildup of some inducer. A starvation for carbon and inorganic phosphate induced mcjA expression, while under nitrogen limitation there was no induction at all. These results taken together suggest that stationary-phase induction of mcjA is triggered by nutrient depletion. The mcjB and mcjC genes were also regulated by the growth phase of the culture, but in contrast to mcjA, they showed substantial expression already during exponential growth. Induction of the microcin genes was demonstrated to be independent of RpoS, the cyclic AMP-Crp complex, OmpR, and H-NS. Instead, we found that the growth-phase-dependent expression of mcjA, mcjB, and mcjC may be explained by the concerted action of the positively acting transition state regulators ppGpp, Lrp, and integration host factor. Measurements of microcin J25 production by strains defective in these global regulators showed a good correlation with the reduced expression of the fusions in such mutant backgrounds. 相似文献
13.
为了减少rIL-2工程菌高密度培养时乙酸的积累,在诱导阶段对该工程菌进行细胞再循环培养的研究,比较了细胞再循环补料液、pH、细胞循环培养时间段对工程菌的生长及rIL-2表达的影响。结果表明在菌密度D_(600)为50时,细胞再循环补料液中酵母抽提物与胰蛋白胨浓度为发酵培养基的5倍就能满足rIL-2表达的需求,同时选择诱导后4~6h之间的细胞再循环培养能有效地防止乙酸的过高积累并减少营养物质的损失,有利于rIL-2的表达。根据以上研究结果得到了rIL-2工程菌诱导阶段细胞再循环培养方法,使得在诱导前菌密度D_(600)为50左右时rIL-2的表达水平约为40%。 相似文献
14.
碳源和IPTG诱导对工程菌MM2表达CTB的影响 总被引:1,自引:0,他引:1
研究了不同碳源如葡萄糖、乳酸和乙酸以及IPTG诱导对工程菌MM2表达霍乱毒素B亚单位(CTB)的影响。在YC培养基中分别加入0.048mol/L的葡萄糖、0.102mol/L的乳酸和0.167mol/L的乙酸,它们在完全氧化后可产生相同的能量。结果表明,加入葡萄糖会大幅度降低ctb基因的表达水平,其原因和培养过程中pH值下降有关;加入乳酸可提高ctb基因表达水平1.15倍,且不抑制菌体生长;加入乙酸可提高ctb基因的表达水平0.9倍,但对菌体生长有抑制作用。不同时间及不同浓度的IPTG诱导未能提高ctb基因的表达水平,说明lac启动子对ctb基因的表达没有影响或影响很小。 相似文献
15.
George HA Powell AL Dahlgren ME Herber WK Maigetter RZ Burgess BW Stirdivant SM Greasham RL 《Biotechnology and bioengineering》1992,40(3):437-445
Physiological effects of isopropyl-thiogalactopyranoside (IPTG) induction were examined in Escherichia coli strain JM109 expressing a fusion protein composed of transforming growth factor alpha and a 40-kD portion of Pseudomonas aeruginosa exotoxin A (TGF(alpha)-PE40) under control of the tac promoter. Fermentations at the 15-L scale in complex medium compared growth and metabolite profiles of the untransformed JM109 host strain, the strain transformed with the vector lacking the TGF(alpha)-PE40 open reading frame (JM109[pKK2.7]), and the strain with the complete plasmid for TGF(alpha)-PE40 expression (JM109[pTAC-TGF57-PE40]). Metabolite and growth profiles of JM109 (pTAC-TGF57-PE40) cultures changed significantly in IPTG-induced versus uninduced cultures. Prior to induction, glucose was metabolized to acetate or completely oxidized to CO(2). Following induction, pyruvate was also excreted in addition to acetate. In the absence of inducer, pyruvate was excreted by JM109 (pTAC-TGF57-PE40) only when dissolved oxygen levels fell to less than 10% of saturation (microaerobic rather than anaerobic conditions). The untransformed JM109 host strain or JM109 (pKK2.7) did not excrete pyruvate in the presence or absence of inducer, although JM109 (pKK2.7) exhibited a pattern of growth following addition of IPTG that closely resembled JM109 (pTAC-TFG57-PE40). Fermentations of JM109 (pTAC-TFG57-PE40) in a synthetic medium supported lower expression levels, but resulted in similar alterations in metabolite profiles. Induction in synthetic medium resulted in pyruvate excretion without further acetate accumulation. Taken together, these data suggest that one consequence of TGF(alpha)-PE40 expression in JM109 is altered patterns of pyruvate oxidation. (c) 1992 John Wiley & Sons, Inc. 相似文献
16.
The primary advantage of an inducible promoter expression system is that production of the recombinant protein can be biochemically controlled, allowing for the separation of unique growth and production phases of the culture. During the growth phase, the culture is rapidly grown to high cell density prior to induction without the extra metabolic burden of exogenous protein production, thus minimizing the nonproductive period of the culture. Induction of the culture at high cell density ensures that the volumetric production will be maximized. In this work, we have demonstrated the feasibility of overexpressing a reporter glycoprotein from the inducible MMTV promoter in recombinant Chinese hamster ovary (CHO) cells cultured in a high cell density perfusion bioreactor system. Retention of suspension-adapted CHO cells was achieved by inclined sedimentation. To maximize volumetric production of the culture, we have demonstrated that high cell density must be achieved prior to induction. This operating scheme resulted in a 10-fold increase in volumetric titer over the low density induction culture, corresponding directly to a 10-fold increase in viable cell density during the highly productive period of the culture. The amount of glycoprotein produced in this high cell density induction culture during 26 days was 84-fold greater than that produced in a week long batch bioreactor. Long-term perfusion cultures of the recombinant cell line showed a production instability, a phenomenon that is currently being investigated. 相似文献
17.
The use of glucose starvation to uncouple the production of recombinant beta-galactosidase from cell growth in Escherichia coli was investigated. A lacZ operon fusion to the carbon starvation-inducible cst-1 locus was used to control beta-galactosidase synthesis. beta-Galactosidase induction was observed only under aerobic starvation conditions, and its expression continued for 6 h following the onset of glucose starvation. The cessation of beta-galactosidase expression closely correlated with the exhaustion of acetate, an overflow metabolite of glucose, from the culture medium. Our results suggest the primary role of acetate in cst-1-controlled protein expression is that of an energy source. Using this information, we metered acetate to a glucose-starved culture and produced a metabolically sluggish state, where growth was limited to a low linear rate and production of recombiant beta-galactosidase occurred continuously throughout the experiment. The cst-1 controlled beta-galactosidase synthesis was also induced at low dilution rates in a glucose-limited chemostat, suggesting possible applications to high-density cell systems such as glucose-limited recycle reactors. This work demonstrates that by using an appropriate promoter system and nutrient limitation, growth can be restrained while recombinant protein production is induced and maintained. 相似文献
18.
Perfusion cultures of recombinant Chinese hamster ovary cells, producing recombinant antibody against the S surface antigen of Hepatitis B virus, were carried out in continuous and intermittent mode using a Centritech Lab II Centrifuge. In the continuous perfusion process, despite the absence of shear stress from the pump head, long-term operation was not possible because of continuously repeated exposure to oxygen limitation and low temperature, as well as shear stress from centrifugal force. In the intermittent perfusion processes, the frequency of cell-passage through the centrifuge was substantially reduced, compared with the continuous perfusion mode; however, the degree of reduction could not guarantee stable long-term operation. Although various operating parameters were applied in the intermittent perfusion cultures, high cell densities could not be maintained stably. In a single bioreactor culture system, a specific cell that is returned from the centrifuge to the bioreactor could be transferred from the bioreactor to the centrifuge again in the next cycle. These repetitive damages, caused by shear stress from the pump head and centrifugal force, as well as exposure to suboptimal conditions such as oxygen limitation and low temperature below 37 degrees C, were more serious at higher perfusion rates. Subsequently, damaged cells and dead cells were continuously accumulated in the bioreactor. Culture temperature shift from 37 to 33 degrees C increased antibody concentrations but showed inhibitory effects on cell growth. The negative effects of lowering culture temperature on cell growth overwhelmed the positive effects on antibody production. To protect cells from shear stress, Pluronic F-68 was 2-fold concentrated in the culture medium; nevertheless, a significantly higher concentration of Pluronic F-68 (2 g/L) may have inhibitory effects on cell growth. 相似文献
19.
Stromal-vascular (S-V) cells from rat inguinal fat depots were isolated and cultured in medium containing fetal bovine serum (FBS) and differentiated in defined medium until lipid accumulation was apparent. C/EBPalpha, beta and delta levels were evaluated for different growth conditions and at different times using Western blots. Immediately after isolation C/EBPalpha, beta and delta could not be detected in S-V cells. After seeding for 24 h in Dulbecco's modified Eagle's medium (DMEM) with FBS, C/EBPalpha, beta and delta could all be detected. Cells at day 1 of culture in insulin, transferrin, triiodothyronine and selenium (ITTS) had increased levels of C/EBPalpha and continued steady high levels to day 6 of culture. Cultures grown in DMEM alone, with no ITTS, showed C/EBPalpha levels similar to ITTS cultures at day 1 and day 3; however, levels diminished after day 3. DMEM cultures also showed lipid accumulation at day 6; however, the number of cells and the amount of lipid cell were reduced from levels observed in ITTS cultures. C/EBPbeta was expressed uniformly throughout the culture period in either DMEM or ITTS cultures while C/EBPdelta expression was higher with DMEM treatment than with ITTS. Treatment of 2 day DMEM cultures with FBS increased levels of C/EBPbeta and delta but significantly reduced levels of C/EBPalpha. Immunocytochemical analysis of S-V cells at day 1 of culture showed a similar percentage of cells stained in DMEM cultures and ITTS cultures. However, by day 6 of culture the percentage of cells staining positively for C/EBPalpha in DMEM had been reduced by one half while in ITTS the percent positive cells remained about the same. Our results indicate that ITTS is not necessary for the induction of C/EBPalpha and accumulation of lipid in S-V cells. However, ITTS is responsible for maintaining C/EBPalpha and enhanced lipid accumulation. Because C/EBPalpha, beta and delta expression occurs very early in cell culture and C/EBPalpha and delta expression continues to increase in DMEM without any apparent inducing agents, our results suggest that these factors may be expressed by the same cells in vivo before being placed in culture. Thus, a large fraction of S-V cells may be further along in the differentiation program than 3T3 cells are when they begin differentiation. 相似文献
20.
Schlatmann JE Moreno PR Vinke JL Ten Hoopen HJ Verpoorte R Heijnen JJ 《Biotechnology and bioengineering》1994,44(4):461-468
Oxygen and nutrient limitation was investigated in order to identify the origin of a lower specific ajmalicine production in Catharanthus roseus cultures at high cell densities in an induction medium. The effect of oxygen limitation was explored by comparing two identically aerated and agitated high cell density bioreactor cultures with dissolved oxygen (DO) concentration of 15% and 85% of air saturation, with respect to alkaloid formation and related enzymes activities. Oxygen had an evident effect on ajmalicine production: in the high DO cultures production was more than 5 times higher than in the low DO cultures. The difference in ajmalicine production between high and low DO could not be explained by the enzyme activity profiles. Moreover, the productivity in the high density culture could not restored to the level of a low density culture (at a high DO) by increasing the DO alone. The effect of nutrient limitation was studied with response surface methodology in shake flask cultures. Nutrient limitation could not be demonstrated to be responsible for the productivity loss. Alkaloid and enzyme measurements in the shake flask cultures supported previous findings that the tryptamine pathway may regulate alkaloid production, provided that the terpenoid pathway is sufficiently active. (c) 1994 John Wiley & Sons, Inc. 相似文献