共查询到20条相似文献,搜索用时 31 毫秒
1.
The recombinant human granulocyte-colony-stimulating factor (rhG-CSF) was synthesized in a fusion protein using a GAL1-10 UAS in recombinant Saccharomyces cerevisiae and the intracellular KEX2 cleavage led excretion of mature rhG-CSF into the extracellular culture broth. The recombinant yeast growth in fed-batch cultures was controlled by precise computer-aided medium feed. The optimal C/N ratio in preinduction (glucose/Casamino acids) and post-induction (galactose/yeast extract) feed media was determined at 3 and 2, respectively. The final rhG-CSF and cell concentration was more than 60 mg/L and 70 g/L, respectively, with around 90% plasmid stability and negligible ethanol accumulation. Comparing the cell growth between the hG-CSF + and hG-CSF - recombinant strains shows that the cloned gene product does not hamper the host cell growth. 相似文献
2.
Babu KR Swaminathan S Marten S Khanna N Rinas U 《Applied microbiology and biotechnology》2000,53(6):655-660
Escherichia coli TG1 transformed with a temperature-regulated interferon-α expression vector was grown to high cell density in defined medium
containing glucose as the sole carbon and energy source, utilizing a simple fed-batch process. Feeding was carried out to
achieve an exponential increase in biomass at growth rates which minimized acetate production. Thermal induction of such high
cell density cultures resulted in the production of ∼4 g interferon-α/l culture broth. Interferon-α was produced exclusively
in the form of insoluble inclusion bodies and was solubilized under denaturing conditions, refolded in the presence of arginine
and purified to near homogeneity, utilizing single-step ion-exchange chromatography on Q-Sepharose. The yield of purified
interferon-α was ∼300 mg/l with respect to the original high cell density culture broth (overall yield of ∼7.5% active interferon-α).
The purified recombinant interferon-α was found by different criteria to be predominantly monomeric and possessed a specific
bioactivity of ∼2.5 × 108 IU/mg based on viral cytopathic assay.
Received: 8 October 1999 / Received revision: 8 December 1999 / Accepted: 12 December 1999 相似文献
3.
4.
C. S. Shin M. S. Hong D. Y. Kim H. C. Shin J. Lee 《Applied microbiology and biotechnology》1998,49(4):364-370
Synthesis of two recombinant proteins (human glucagon and human growth hormone) was investigated in fed-batch cultures at
high cell concentrations of recombinant Escherichia coli. The glucose-limited growth was achieved without accumulation of metabolic by-products and hence the cellular environment
is presumed invariable during growth and recombinant protein synthesis. Via exponential feeding in the two-phase fed-batch
operation, the specific cell growth rate was successfully controlled at the desired rates and the fed-batch mode employed
is considered appropriate for examining the correlation between the specific growth rate and the efficiency of recombinant
product formation in the recombinant E. coli strains. The two recombinant proteins were expressed as fusion proteins and the concentration in the culture broth was increased
to 15 g fusion growth hormone l−1 and 7 g fusion glucagon l−1. The fusion growth hormone was initially expressed as soluble protein but seemed to be gradually aggregated into inclusion
bodies as the expression level increased, whereas the synthesized fusion glucagon existed as a cytoplasmic soluble protein
during the whole induction period. The stressful conditions of cultivation employed (i.e. high-cell-density cultivation at
low growth rate) may induce the increased production of various host-derived chaperones and thereby enhance the folding efficiency
of synthesized heterologous proteins. The synthesis of the recombinant fusion proteins was strongly growth-dependent and more
efficient at a higher specific growth rate. The mechanism linking specific growth rate with recombinant protein productivity
is likely to be related to the change in cellular ribosomal content.
Received: 27 May 1997 / Received last revision: 31 October 1997 / Accepted: 21 November 1997 相似文献
5.
Kinetic models for astaxanthin production by high cell density mixotrophic culture of the microalga Haematococcus pluvialis 总被引:3,自引:0,他引:3
High cell density cultivation of Haematococcus pluvialis for astaxanthin production was carried out in batch and fed-batch modes in 3.7-L bioreactors with stepwise increased light
intensity control mode. A high cell density of 2.65 g L−1 (batch culture) or 2.74 g L−1 (fed-batch culture) was obtained, and total astaxanthin production in the fed-batch culture (64.36 mg L−1) was about 20.5% higher than in the batch culture (53.43 mg L−1). An unstructured kinetic model to describe the microalga culture system including cell growth, astaxanthin formation, as
well as sodium acetate consumption was proposed. Good agreement was found between the model predictions and experimental data.
The models demonstrated that the optimal light intensity for mixotrophic growth of H. pluvialis in batch or fed-batch cultures in a 3.7-L bioreactor was 90–360 μmol m−2 s−1, and that the stepwise increased light intensity mode could be replaced by a constant light intensity mode.
Received 24 December 1998/ Accepted in revised form 23 April 1999 相似文献
6.
B H Chung Y J Choi S H Yoon S Y Lee Y I Lee 《Journal of industrial microbiology & biotechnology》2000,24(2):94-99
Fed-batch cultures were carried out to overproduce human insulin-like growth factor I (IGF-I) in Escherichia coli. The effects of carbon sources (glucose or glycerol) and induction time on cell growth and IGF-I production were investigated
in more detail. Glycerol was a better carbon source than glucose for IGF-I production in fed-batch culture. Induction at the
mid-exponential phase with glycerol as a carbon source in the pH-stat fed-batch culture was optimal for IGF-I production.
Under this condition, 2.8 g L−1 of fusion IGF-I was produced as inclusion bodies. We have also developed downstream processing for preparative scale purification
of IGF-I from the fusion protein produced by the fed-batch culture using glycerol as a carbon source. After the fusion protein
expressed was solubilized in 8 M urea and cleaved with hydroxylamine, the released IGF-I was purified by cation exchange chromatography,
refolding and preparative scale reverse phase HPLC (rp-HPLC) to give recombinant IGF-I of >98% purity. The biological activities
of the purified IGF-I were measured and found to be identical to those of commercial IGF-I. Journal of Industrial Microbiology & Biotechnology (2000) 24, 94–99.
Received 13 January 1999/ Accepted in revised form 02 October 1999 相似文献
7.
In this work, the effect of the feeding strategy in Zymomonas mobilis CP4 fed-batch fermentations on the final biomass and ethanol concentrations was studied. Highest glucose yields to biomass
(0.018 g/g) and to ethanol (0.188 g/g) were obtained in fed-batch fermentations carried out using different feeding rates
with a glucose concentration in the feed equal to 100 g/l. Lower values (0.0102 g biomass/g glucose and 0.085 g ethanol/g
glucose) were obtained when glucose accumulated to levels higher than 60 g/l. On the other hand, the highest biomass (5 g/l)
and ethanol (39 g/l) concentrations were obtained using a glucose concentration in the feed equal to 220 g/l and exponentially
varied feeding rates. Experimental data were used to validate the mathematical model of the system. The prediction errors
of the model are 0.39, 14.36 and 3.24 g/l for the biomass, glucose and ethanol concentrations, respectively. Due to the complex
relationship for describing the specific growth rate, a fed-batch culture in which glucose concentration is constant would
not optimize the process.
Received: 30 November 1999 / Received revision: 24 March 2000 / Accepted: 7 April 2000 相似文献
8.
Dilsen S Paul W Sandgathe A Tippe D Freudl R Thömmes J Kula MR Takors R Wandrey C Weuster-Botz D 《Applied microbiology and biotechnology》2000,54(3):361-369
A pH-auxostatic fed-batch process was developed for the secretory production of a fusion protein consisting of the pro-part
of Staphylococcus hyicus lipase and two synthetic human calcitonin (hCT) precursor repeats under the control of a xylose-inducible promotor from Staphylococcus xylosus. Using glycerol as the energy source and pH-controlled addition of yeast extract resulted in the production of 2000 mg l−1 of the fusion protein (420 mg l−1 of the recombinant hCT precursor) within 14 h, reaching 45 g l−1 cell dry mass with Staphylococcus carnosus in a stirred-tank reactor. Product titer and space-time yield (30 mg calcitonin precursor l−1 h−1) were thus improved by a factor of 2, and 4.5, respectively, compared to Escherichia coli expression-secretion systems for the production of calcitonin precursors. Two hundred grams of the fusion protein was secreted
by the recombinant S. carnosus on a 150-l scale (scale-up factor of 50) with a minimum use of technical-grade yeast extract (40 mg fusion protein g−1 yeast extract).
Received: 18 January 2000 / Received revision: 14 April 2000 / Accepted: 14 April 2000 相似文献
9.
Ashkenazy R Yannai S Rahman R Rabinovitz E Gottlieb L 《Applied microbiology and biotechnology》1999,52(4):608-611
Spent Saccharomyces cerevisiae cells from a beer fermentation process were evaluated for lead cation sorption. The crude biomass was washed with water and
acetone prior to any other treatment. Although the washed biomass showed substantial lead ion sorption it was susceptible
to microbial spoilage. Different aldehydes were tested as chemical fixation agents; however, most of them caused drastic lowering
of the metal uptake capacity. However, benzaldehyde was not only an excellent fixation agent, but the biomass treated with
it also retained its original lead sorption capacity. A mechanism for the fixation process is suggested.
Received: 11 January 1999 / Received revision: 26 April 1999 / Accepted: 1 May 1999 相似文献
10.
J Lin D Panigraphy L B Trinh J Folkman J Shiloach 《Journal of industrial microbiology & biotechnology》2000,24(1):31-35
A pilot-scale production method of recombinant human angiostatin, a 38-kD fragment of plasminogen which has been reported
to have antiangiogenic activity, has been successfully established by expressing the protein in the methylotrophic yeast Pichia pastoris. The secreted protein inhibited cultured endothelial cell proliferation in vitro and Lewis lung carcinoma growth in mice. The fermentation process was carried out using an on-line methanol controller, administering
methanol to the growing culture and keeping its concentration under 2 g L−1. The fermentation lasted 90 h, of which 70 h were growth on methanol. During growth on methanol the culture volume increased
64%, from 7 L to 11.5 L, producing 200 mg angiostatin and 5 kg of biomass. Journal of Industrial Microbiology & Biotechnology (2000) 24, 31–35.
Received 12 May 1999/ Accepted in revised form 06 September 1999 相似文献
11.
C. A. Dunster K. H. Cheeseman S. P. Maddix 《Applied microbiology and biotechnology》1997,48(2):198-203
The CHO320 cell line, engineered to produce human interferon γ was investigated with regard to its susceptibility to oxidative
stress. Batch cultures of the cells grown in a bench-top bioreactor exhibited no marked response to changes in oxygen concentration
between 6% and 14% whereas cell growth and recombinant protein production were inhibited by increasing the oxygen to 20%.
High concentrations of hydrogen peroxide (in excess of 200 μM) were required to inhibit growth of the CHO320 cells whereas
concentrations of 50 μm and 100 μM had no effect on recombinant protein production. Buthionine sulphoximine (50 μM and 100 μM)
completely depleted the cells of glutathione within 24 h; however, no quantitative effect on recombinant protein production
was seen. It is concluded that the CHO320 cells are, possibly as a consequence of the long selection process they have undergone,
very resistant to oxidative stress.
Received: 14 November 1996 / Received last revision: 14 April 1997 / Accepted: 19 April 1997 相似文献
12.
Jeffke T Jende D Mätje C Ehlers RU Berthe-Corti L 《Applied microbiology and biotechnology》2000,54(3):326-330
Photorhabdus luminescens, a bacterial symbiont of entomopathogenic biocontrol nematodes, was grown in batch and glucose fed-batch culture. The cell
density, bioluminescence, production of antibiotic substances, number of cells with inclusion bodies, glucose concentration
and oxygen uptake rate were recorded. The addition of 12.4 g l−1 glucose prolonged the growth, and the yield almost doubled, from 6.85 g l−1 to 12.45 g l−1 dry mass. The production of antibiotic substances increased by 140%. Bioluminescence was higher in the batch culture. A shift
of P. luminescens to phase II variants was not detected.
Received: 21 January 2000 / Received revision: 3 April 2000 / Accepted: 7 April 2000 相似文献
13.
New secretion vectors containing the Bacillus sp. endoxylanase signal sequence were constructed for the secretory production of recombinant proteins in Escherichia coli. The E. coli alkaline phosphatase structural gene fused to the endoxylanase signal sequence was expressed from the trc promoter in various E. coli strains by induction with IPTG. Among those tested, E. coli HB101 showed the highest efficiency of secretion (up to 25.3% of total proteins). When cells were induced with 1 mM IPTG,
most of the secreted alkaline phosphatase formed inclusion bodies in the periplasm. However, alkaline phosphatase could be
produced as a soluble form without reduction of expression level by inducing with less (0.01 mM) IPTG, and greater than 90%
of alkaline phosphatase could be recovered from the periplasm by the simple osmotic shock method. Fed-batch cultures were
carried out to examine the possibility of secretory protein production at high cell density. Up to 5.2 g/l soluble alkaline
phosphatase could be produced in the periplasm by the pH-stat fed-batch cultivation of E. coli HB101 harboring pTrcS1PhoA. These results demonstrate the possibility of efficient secretory production of recombinant proteins
in E. coli by high cell density cultivation.
Received: 8 September 1999 / Received revision: 3 January 2000 / Accepted 4 January 2000 相似文献
14.
Khalilzadeh R Mohammadian-Mosaabadi J Bahrami A Nazak-Tabbar A Nasiri-Khalili MA Amouheidari A 《Journal of industrial microbiology & biotechnology》2008,35(12):1643-1650
The fed-batch process using glucose as the sole source of carbon and energy with exponential feeding rate was carried out
for high cell density cultivation of recombinant Escherichia coli BL21 (DE3) expressing human granulocyte-colony stimulating factor (hG-CSF). IPTG was used to induce the expression of hG-CSF
at 48 g dry cell wt l−1 during high cell density culture of recombinant E. coli BL21 (DE3) [pET23a-g-csf]. The final cell density, specific yield and overall productivity of hG-CSF were obtained as ~64 g
dry cell wt l−1, 223 mg hG-CSF g−1 dry cell wt and 775 mg hG-CSF l−1 h−1, respectively. The resulting purification process used cell lysis, inclusion body (IB) preparation, refolding, DEAE and Butyl-Sepharose.
Effects of different process conditions such as cell lysis and washing of IB were evaluated. The results reveal that the cells
lyzed at 1,200 bar, 99.9% and Triton removed about 64% of the LPS but sarcosyl had no effect on removal of nucleic acids and
LPS. Further analysis show that DEAE column removes DNA about 84%. Cupper concentration was identified as parameter that could
have a significant impact on aggregation, as an unacceptable pharmaceutical form that decrease process yields. The purity
of purified hG-CSF was more than 99%. Also the comparison of activity between purified hG-CSF and commercial form do not show
valuable decrease in activity in purified form. 相似文献
15.
Phycocyanin production by high cell density cultivation of Spirulina platensis in batch and fed-batch modes in 3.7-L bioreactors with a programmed stepwise increase in light intensity program was investigated.
The results showed that the cell density in fed-batch culture (10.2 g L−1) was 4.29-fold that in batch culture (2.38 g L−1), and the total phycocyanin production in the fed-batch culture (0.795 g L−1) was 3.05-fold that in the batch culture (0.261 g L−1). An unstructured kinetic model to describe the microalga culture system including cell growth, phycocyanin formation, as
well as glucose consumption was proposed. The data fitted the models well (r
2 > 0.99). Furthermore, based on the kinetic models, the potential effects of light limitation and photoinhibition on cell
growth and phycocyanin formation can be examined in depth. The models demonstrated that the optimal light intensity for mixotrophic
growth of Spirulina platensis in batch or fed-batch cultures using a 3.7-L bioreactor was 80160 μE m−2 s−1, and the stepwise increase in light intensity can be replaced by a constant light intensity mode.
Received 28 July 1998/ Accepted in revised form 8 October 1998 相似文献
16.
Poly-(3-hydroxybutyrate) production from whey by high-density cultivation of recombinant Escherichia coli 总被引:2,自引:0,他引:2
Recombinant Escherichia coli strain GCSC 6576, harboring a high-copy-number plasmid containing the Ralstonia eutropha genes for polyhydroxyalkanoate (PHA) synthesis and the E. coli ftsZ gene, was employed to produce poly-(3-hydroxybutyrate) (PHB) from whey. pH-stat fed-batch fermentation, using whey powder
as the nutrient feed, produced cellular dry weight and PHB concentrations of 109 g l−1 and 50 g l−1 respectively in 47 h. When concentrated whey solution containing 210 g l−1 lactose was used as the nutrient feed, cellular dry weight and PHB concentrations of 87 g l−1 and 69 g l−1 respectively could be obtained in 49 h by pH-stat fed-batch culture. The PHB content was as high as 80% of the cellular dry
weight. These results suggest that cost-effective production of PHB is possible by fed-batch culture of recombinant E. coli using concentrated whey solution as a substrate.
Received: 19 December 1997 / Received revision: 17 March 1998 / Accepted: 20 March 1998 相似文献
17.
A cDNA fragment encoding the A catalytic domain of the Neocallimastix frontalis endoxylanase XYN3 was amplified and cloned by the polymerase chain reaction technique. The xyn3A DNA fragment was inserted between the Saccharomyces cerevisiae phosphoglycerate kinase gene promoter and terminator sequences on a multicopy episomal plasmid for Kluyveromyces lactis. The XYN3A domain was successfully expressed in K. lactis and functional endoxylanase was secreted by the yeast cells with the K. lactis killer toxin secretion signal. The XYN3A domain was also expressed in a strain of Penicillium roqueforti as a fusion protein (ShBLE::XYN3A) of the phleomycin-resistance gene product and the endoxylanase. Active endoxylanase was
efficiently secreted from the fungal cells with the Trichoderma viride cellobiohydrolase (CBH1) secretion signal and processed by a related KEX2 endoprotease of the secretion pathway. Several
differently glycosylated forms of the recombinant enzymes were secreted by the yeast and the filamentous fungus.
Received: 10 November 1998 / Received revision: 8 March 1999 / Accepted: 14 March 1999 相似文献
18.
Terashima M Murai Y Kawamura M Nakanishi S Stoltz T Chen L Drohan W Rodriguez RL Katoh S 《Applied microbiology and biotechnology》1999,52(4):516-523
Recombinant human α1-antitrypsin (rAAT) was expressed and secreted from transgenic rice cell suspension cultures in its biologically active form.
This was accomplished by transforming rice callus tissues with an expression vector, p3D-AAT, containing the cDNA for mature
human AAT protein. Regulated expression and secretion of rAAT from this vector was achieved using the promoter, signal peptide,
and terminator from a rice α-amylase gene Amy3D. The Amy3D gene of rice is tightly controlled by simple sugars such as sucrose. It was possible, therefore, to induce the expression
of the rAAT by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically.
Although transgenic rice cell produced a heterogeneous population of the rAAT molecules, they had the same N-terminal amino
acids as those found in serum-derived (native) AAT from humans. This result indicates that the rice signal peptidase recognizes
and cleaves the novel sequence between the Amy3D signal peptide and the first amino acid of the mature human AAT. The highest molecular weight band seen on Western blots
(AAT top band) was found to have the correct C-terminal amino acid sequence and normal elastase binding activity. Staining
with biotin-concanavalin A and avidin horseradish peroxidase confirmed the glycosylation of the rAAT, albeit to a lesser extent
than that observed with native AAT. The rAAT, purified by immunoaffinity chromatography, had the same association rate constant
for porcine pancreatic elastase as the native AAT. Thermostability studies revealed that the rAAT and native AAT decayed at
the same rate, suggesting that the rAAT is correctly folded. The productivity of rice suspension cells expressing rAAT was
4.6–5.7 mg/g dry cell. Taken together, these results support the use of rice cell culture as a promising new expression system
for production of biologically active recombinant proteins.
Received: 18 January 1999 / Received revision: 26 April 1999 / Accepted: 1 May 1999 相似文献
19.
Cserjan-Puschmann M Kramer W Duerrschmid E Striedner G Bayer K 《Applied microbiology and biotechnology》1999,53(1):43-50
The aim of this work was the establishment of a novel method to determine the metabolic load on host-cell metabolism resulting
from recombinant protein production in Escherichia coli. This tool can be used to develop strategies to optimise recombinant fermentation processes through adjustment of recombinant-protein
expression to the biosynthetic capacity of the host-cell. The signal molecule of the stringent-response network, guanosine
tetraphosphate (ppGpp), and its precursor nucleotides were selected for the estimation of the metabolic load relating to recombinant-protein
production. An improved analytical method for the quantification of nucleotides by ion-pair, high-performance liquid chromatography
was established. The host-cell response upon overexpression of recombinant protein in fed-batch fermentations was investigated
using the production of human superoxide dismutase (rhSOD) as a model system. E. coli strains with different recombinant systems (the T7 and pKK promoter system) exerting different loads on host-cell metabolism
were analysed with regard to intracellular nucleotide concentration, rate of product formation and plasmid copy number.
Received: 30 April 1999 / Received revision: 26 July 1999 / Accepted: 1 August 1999 相似文献
20.
The effect of different aeration conditions during the culture of Azotobacter vinelandii on the production and molecular mass of alginate was evaluated in shake flasks. In baffled flasks, the bacteria grew faster
and produced less alginate (1.5 g/l) than in conventional (unbaffled) flasks (4.5 g/l). The viscosity of the culture broth
was also influenced by the type of flask. Higher final viscosities were attained in unbaffled flasks [520 cP (520 mPa s)]
as compared to baffled flasks (30 cP). This latter phenomenon was closely related to the changes in the molecular mass distribution.
In either cases, the mean molecular mass increased with culture age; however, at the end of the fermentation, the mean molecular
mass of the alginate obtained in unbaffled flasks was fivefold higher than that obtained in baffled flasks. As the culture
proceeded, the cells of Azotobacter grown in unbaffled flasks increased in diameter, whereas those cultured in baffled flasks decreased in size.
Received: 13 December 1996 / Received revision: 10 April 1997 / Accepted: 27 April 1997 相似文献