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1.
J L Schwartz E B Ferrari J Terracciano J Troyanovich I Gunnarsson J Wright-Minogue J W Chen A D Kwong 《Journal of industrial microbiology & biotechnology》1997,19(2):87-91
A gene expression system using recombinant Autographa californica nuclear polyhedrosis virus (baculovirus) and Sf-9 cells has been scaled up to the 10-L tank level and shown to be capable
of producing herpes simplex virus (HSV) protease in serum-free media. High densities of Spodoptera frugiperda (Sf-9) cells were achieved by modifying two 10-L Biolafitte fermenters specifically for insect cell growth. The existing
Rushton impellers were replaced by marine impellers to reduce shear and the aeration system was modified to allow external
addition of air/O2 mixtures at low flow rates through either the sparge line or into the head space of the fermenter. To inoculate the tanks,
Sf-9 cells were adapted to grow to high cell densities (6–10 × 106 cells ml−1) in shake flasks in serum-free media. With these procedures, cell densities of 5 × 106 cells ml−1 were routinely achieved in the 10-L tanks. These cells were readily infected with recombinant baculovirus expressing the
247-amino acid catalytic domain of the HSV-1 strain 17 protease UL26 gene as a glutathione-S-transferase (GST) fusion protein (GST-247). Three days after infection at a multiplicity of infection (MOI) of 3 pfu cell−1, the GST-247 fusion protein was purified from a cytoplasmic lysate by Glutathione Sepharose 4-B affinity chromatography
with reproducible yields of 11–38 mg L−1 of recombinant protein and ≥ 90% purity. Maximum production of this protein was observed at a cell density of 5.0 × 106 cells ml−1.
Received 09 December 1996/ Accepted in revised form 13 April 1997 相似文献
2.
Large scale transient 5-HT3 receptor production with the Semliki Forest Virus Expression System 总被引:1,自引:0,他引:1
H. D. Blasey B. Brethon R. Hovius H. Vogel H A. P. Tairi K. Lundström L. Rey A. R. Bernard 《Cytotechnology》2000,32(3):199-208
The expression of recombinant proteins with the Semliki Forest Virus (SFV) system has been scaled up to bioreactor scale. As a model protein for this study the human 5-HT3 receptor was chosen. The gene for the receptor was subcloned into the SFV expression plasmid pSFV1. Virus production by in vivo packaging and production of the recombinant protein was scaled up, the latter to a reactor volume of 11.5 l. A VibromixTM agitation system was chosen to overcome aggregation problems of BHK cells in suspension. In the process, cells were first grown to a density of 106 cells/ml, the medium was then exchanged with fresh medium and the culture was infected with the recombinant virus at an estimated multiplicity of infection of 30. 24 h post infection we measured an expression level of 3 million functional 5-HT3 receptors per cell. For harvesting, the cells were pelleted by centrifugation. The receptor protein was purified in a single step (Hovius et al., 1998) by exploiting the hexa-His tag at minimal protein loss (51% yield). Experiments to optimise expression resulted in yields up to 8 million receptors per cell, when the pH of a suspension culture was controlled at pH 7.3. Rapid virus generation and protein production, high protein yields as well as successful large scale application have made the SFV expression system attractive to produce large quantities of recombinant protein in a very short time. After optimisation of the expression conditions (in particular by setting the pH at 7.3), yields were increased twofold. 相似文献
3.
The baculovirus expression vector system was employed to produce human apolipoprotein E and β-galactosidase in order to study
the effect of multiplicity of infection on secreted and non-secreted recombinant protein production. Prior knowledge of the
influence of other cell culture and infection parameters, such as the cell density at time of infection and the time of harvest,
allowed determination of the direct and indirect influences of multiplicity of infection on recombinant protein synthesis
and degradation in insect cells. Under non-limited, controlled conditions, the direct effect of multiplicity of infection
(10−1−10 pfu/cell) on specific recombinant product yields of non-secreted β-galactosidase was found to be insignificant. Instead,
the observed increased in accumulated product was directly correlated to the total number of infected cells during the production
period and therefore ultimately dependent on an adequate supply of nutrients. Only the timing of recombinant virus and protein
production was influenced by, and dependent on the multiplicity of infection. Evidence is presented in this study that indicates
the extremely limited predictability of post-infection cell growth at very low multiplicities of infection of less than 0.1
pfu/cell. Due to the inaccuracy of the current virus quantification techniques, combined with the sensitivity of post-infection
cell growth at low MOI, the possibility of excessive post-infection cell growth and subsequent nutrient limitation was found
to be significantly increased. Finally, as an example, the degree of product stability and cellular and viral protein contamination
at low multiplicity of infection is investigated for a secreted recombinant form of human apolipoprotein E. Comparison of
human apolipoprotein E production and secretion at multiplicities of infection of 10−4−10 pfu/cell revealed increased product degradation and contamination with intracellular proteins at low multiplicities of
infection.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
4.
A novel, disposable-bag bioreactor system that uses wave action for mixing and transferring oxygen was evaluated for BHK 21
C13 cell line growth and Aujeszky’s disease virus (ADV) production. Growth kinetics of BHK 21 C13 cells in the wave bioreactor
during 3-day period were determined. At the end of the 3-day culture period and cell density of 1.82 × 106 cells ml-1, the reactor was inoculated with 9 ml of gE- Bartha K-61 strain ADV suspension (105.9 TCID50) with multiplicity of infection (MOI) of 0.01. After a 144 h incubation period, 400 ml of ADV harvest was obtained with titre
of 107.0 TCID50 ml−1, which corresponds to 40,000 doses of vaccine against AD. In conclusion, the results obtained with the wave bioreactor using
BHK 21 C13 cells showed that this system can be considered as suitable for ADV or BHK 21 C13 cell biomass production. 相似文献
5.
A novel alkylsulfatase gene, sdsAP, was cloned from a newly isolated bacterium Pseudomonas sp. S9. It encoded a protein of 675 amino acids with a calculated molecular mass of 74.9 kDa. The protein contained a typical
N-terminal signal peptide of 41 amino acid residues, followed by a metallo-β-lactamase like domain at the N-terminus and a
SCP-2-like domain at the C-terminus. This domain organization mode suggested that it belonged to the type III sulfatase. The
mature alkylsulfatase was overexpressed in Escherichia coli. The optimal temperature and pH of the recombinant SdsAP were 70°C and 9.0, respectively. Notably, at optimal conditions,
the purified recombinant SdsAP had a high specific activity of 23.25 μmol min−1 mg−1, a K
m (app) of 264.3 μmol, and a V
max (app) of 33.8 μmol min−1 mg−1 for SDS. Additionally, it still retained more than 90% activity after incubation at 65°C for 1 h, which was much different
from other alkylsulfatases reported. The recombinant enzyme hydrolyzed the primary alkyl sulfate such as sodium octyl sulfate
and sodium dodecyl sulfate (SDS). It was a Zn2+-containing and Ca2+ activated alkylsulfatase. This is the first report to explore the various characteristics of the heterologous recombinant
alkylsulfatase in details. These favorable properties could make SdsAP attractive to be useful in the degradation of SDS-containing
waste. 相似文献
6.
Wada T Imanishi T Kawaguchi A Mori MX Mori Y Imoto K Ichida S 《Neurochemical research》2005,30(8):1045-1054
Characteristics for the specific binding of 125I-ω-CTX GVIA and 125I-ω-CTX MVIIC to crude membranes from BHKN101 cells expressing the α1B subunits of Cav2.2 channels and from mice brain lacking the α1B subunits of Cav2.2 channels, particularly, the effects of CaM and various Ca2+ channel blockers on these specific bindings were investigated. Specific binding of 125I-ω-CTX GVIA to the crude membranes from BHKN101 cells was observed, but not from control BHK6 cells. ω-CTX GVIA, ω-CTX MVIIC
and ω-CTX SVIB inhibited the specific binding of 125I-ω-CTX GVIA to crude membranes from BHKN101 cells, and the IC50 values for ω-CTXGVIA, ω-CTX MVIIC and ω-CTX SVIB were 0.07, 8.5 and 1.7 nM, respectively. However, ω-agatoxin IVA and calciseptine
at concentrations of 10−9–10−6 M did not inhibit specific binding. Specific binding was also about 80% inhibited by 20 μg protein/ml CaM. The amount of
125I-ω-CTX GVIA (30 pM) specifically bound to membranes from brain of knockout mice lacking α1B subunits of Cav2.2 channels was about 30% of that to the crude membranes from brain of wild-type. On the other hand, specific binding of
125I-ω-CTX MVIIC (200 pM) was observed on the crude membranes of both BHKN101 and control BHK6 cells. The specific binding of
125I-ω-CTX MVIIC (200 pM) was not inhibited by ω-CTX GVIA and ω-CTX SVIB, and also ω-Aga IVA and calciseptine at concentrations
of 10−9–10−7 M, although specific binding was almost completely dose dependently inhibited by non-radiolabeled ω-CTX MVIIC (IC50 value was about 0.1 nM). 20 μg protein/ml CaM did not inhibit specific binding. Therefore, these results suggest that BHKN101
cells have a typical Cav2.2 channels which are also inhibited by CaM and have not specific binding sites for ω-CTX MVIIC, although ω-CTX MVIIC is
a blocker for both Cav2.1 (α1A; P/Q-type) and Cav2.2 channels. 相似文献
7.
A two-stage continuous culture of Escherichia coli in combination with a bacteriophage λ system was performed in order to overcome the intrinsic plasmid instability that is
frequently observed in recombinant fermentation. A phage λ vector with a Q
− mutation was used to enhance the expression of the λ system. The optimal values of the important operational variables such
as the substrate concentration, the dilution rate, and the mean residence time on the expression of the cloned gene were determined
in both batch and continuous cultures. For all culturing modes, the full induction of the cloned gene was observed 4 h after
the temperature shift. In the two stage continuous culture, the overproduction reached their maxima at D=0.25 h−1 with 1.5 S
0 of the medium supply. The maximum productivity of the total β-galactosidase was 16.3×106 U l−1 h−1, which was approximately seven times higher than that in the single-copy lysogenic stage. The recombinant cells were stable
in the lysogenic state for more than 260 h, while they were stable for 40 h in the lytic state. The instability that developed
rapidly in the second tank is believed to be due to the accumulation of lysis proteins as a result of vector leakage during
the operation. 相似文献
8.
S. Thangminlal Vaiphei Gaurav Pandey K. J. Mukherjee 《Journal of industrial microbiology & biotechnology》2009,36(12):1453-1458
A series of continuous cultures was performed to understand the product formation kinetics of recombinant human interferon
gamma (rhIFN-γ) in Escherichia coli at different dilution rates ranging from 0.1 to 0.3 h−1 in different media. A T7 promoter-based vector was used for expression of IFN-γ in E. coli BL21 (DE3) cells. The recombinant protein was produced as inclusion bodies, thus allowing a rapid buildup of rhIFN-γ inside
the cell, with the specific product yield (Y
p/X
) reaching a maximum value of 182 mg g−1 dry cell weight (DCW). In all the media tested, the specific product formation rate (q
p
) was found to be strongly correlated with the specific growth rate (μ), demonstrating the growth-associated nature of product formation. The q
p
values show no significant decline with time postinduction, even though the recombinant protein has been over produced inside
the cell. The maximum q
p
level of 75.5 mg g−1 h−1 was achieved at the first hour of induction at the dilution rate of 0.3 h−1. Also, this correlation between q
p
and μ was not critically dependent on media composition, which would made it possible to grow cells in defined media in the growth
phase and then push up the specific growth rate just before induction by pulse addition of glucose and yeast extract. This
would ensure the twin objectives of high biomass and high specific productivities, leading to high volumetric product concentration. 相似文献
9.
S. Schöniger T. Caprile C. R. Yulis Q. Zhang E. M. Rodríguez F. Nürnberger 《Cell and tissue research》2009,336(3):477-488
Elevated glutamate levels have been reported in humans with diabetic retinopathy. Retinal Müller glial cells regulate glutamate
levels via the GLAST transporter and system xc
− (cystine-glutamate exchanger). We have investigated whether transporter function and gene and/or protein expression are altered
in mouse Müller cells cultured under conditions of hyperglycemia or oxidative stress (two factors implicated in diabetic retinopathy).
Cells were subjected to hyperglycemic conditions (35 mM glucose) over an 8-day period or to oxidative stress conditions (induced
by exposure to various concentrations of xanthine:xanthine oxidase) for 6 h. The Na+-dependent and –independent uptake of [3H] glutamate was assessed as a measure of GLAST and system xc
− function, respectively. Hyperglycemia did not alter the uptake of [3H] glutamate by GLAST or system xc
−; neither gene nor protein expression decreased. Oxidative stress (70:14 or 100:20 μM xanthine:mU/ml xanthine oxidase) decreased
GLAST activity by ~10% but increased system xc
− activity by 43% and 89%, respectively. Kinetic analysis showed an oxidative-stress-induced change in Vmax, but not Km. Oxidative stress caused a 2.4-fold increase in mRNA encoding xCT, the unique component of system xc
−. Of the two isoforms of xCT (40 and 50 kDa), oxidative stress induced a 3.6-fold increase in the 40-kDa form localized to
the plasma membrane. This is the first report of the differential expression and localization of xCT isoforms as caused by
cellular stress. Increased system xc
− activity in Müller cells subjected to conditions associated with diabetic retinopathy may be beneficial, as this exchanger
is important for the synthesis of the antioxidant glutathione.
This work was supported by NIH R01 EY014560. 相似文献
10.
A Chinese Hamster Ovary cell line, CHO1-15500, producing recombinant human tissue type plasminogen activator (tPA) via the dihydrofolate reductase (DHFR) amplification
system, was studied in batch culture. In this system both DHFR and tPA are under the control of the strong constitutive viral
SV40 early promoter. Employing the cumulative viable cell-hour approach, the specific productivity of tPA had maxima in the
lag (0.065 pg cell−1 h−1) and early decline (0.040 pg cell−1 h−1) population growth phases. The viable population was assigned into four subpopulations (G1, S, G2/M phase, and Apoptotic
cells) using flow cytometric analysis. As expected, intracellular DHFR was maximally expressed during the S cell cycle phase.
The production of tPA, however, was found to be a direct linear function of the G1 phase, with a subpopulation specific productivity
of 0.080 pg c-h−1. Productivity maxima in the lag and early decline corroborate the flow cytometric data, indicative that this recombinant
tPA production occurs primarily in the G1 cell cycle phase, not the S phase. This suggests that endogenous regulatory mechanisms
are important controlling influences on the production of recombinant tPA in this ovarian cell line. Productivity from recombinant
cell lines cannot be inferred from either the plasmid construct or the host cell alone. Elucidation of the relationship between
expression of recombinant protein and the cell cycle phases of the host cell is a major component of the characterization
of the animal cell production system. This information facilitates rational process design, including operating mode, modelling
and control, and medium formulation. 相似文献
11.
Studies were conducted to characterize the effect of gene amplification and foreign gene expression on recombinant CHO cell
growth. Chinese hamster ovary (CHO) cells were transfected with an expression vector containing the gene for dihydrofolate
reductase (dhfr) and the gene for human β-interferon (β-IFN) or thelac Z gene which codes for β-galactosidase (β-gal). The recombinant genes in these CHO cells were amplified stepwise by growth
in 0, 10−7, and 10−6 M methotrexate (MTX), and the β-gal expressing cells were adapted to suspension culture. Flow cytometric methods (FCM) were
used to measure the distribution of amplifieddhfr gene content and foreign β-gal gene expression in the cell populations. A biochemical assay for β-gal was also used. Beta-gal
expression was found to increase with increasing gene amplification. The growth rate of recombinant CHO cells at 10−7 M MTX was found to be 20% lower than that of recombinant CHO cells in MTX-free medium, and the cell growth rate at 10−6 M MTX was 20% lower than that of recombinant CHO cells at 10−7 M MTX. There was no effect of 10−5 M MTX on the growth of CHO-DG44 (dhfr-) cells. The reduction of growth rate in recombinant CHO cells is therefore thought to be mainly due to the effect ofdhfr and foreign gene amplification and increased β-galactosidase expression. 相似文献
12.
Citric acid production from sucrose using a recombinant strain of the yeast Yarrowia lipolytica 总被引:1,自引:0,他引:1
Förster A Aurich A Mauersberger S Barth G 《Applied microbiology and biotechnology》2007,75(6):1409-1417
The yeast Yarrowia lipolytica is able to secrete high amounts of several organic acids under conditions of growth limitation and carbon source excess.
Here we report the production of citric acid (CA) in a fed-batch cultivation process on sucrose using the recombinant Y. lipolytica strain H222-S4(p67ICL1) T5, harbouring the invertase encoding ScSUC2 gene of Saccharomyces cerevisiae under the inducible XPR2 promoter control and multiple ICL1 copies (10–15). The pH-dependent expression of invertase was low at pH 5.0 and was identified as limiting factor of the CA-production
bioprocess. The invertase expression was sufficiently enhanced at pH 6.0–6.8 and resulted in production of 127–140 g l−1 CA with a yield Y
CA of 0.75–0.82 g g−1, whereas at pH 5.0, 87 g l −1 with a yield Y
CA of 0.51 gg−1 were produced. The CA-productivity Q
CA increased from 0.40 g l −1 h−1 at pH 5.0 up to 0.73 g l −1 h−1 at pH 6.8. Accumulation of glucose and fructose at high invertase expression level at pH 6.8 indicated a limitation of CA
production by sugar uptake. The strain H222-S4(p67ICL1) T5 also exhibited a gene–dose-dependent high isocitrate lyase expression
resulting in strong reduction (<5%) of isocitric acid, a by-product during CA production. 相似文献
13.
《Journal of receptor and signal transduction research》2013,33(1-3):115-126
AbstractTwo types of ligand-gated ion channels were expressed with the Semliki Forest virus (SFV) expression system.The cDNAs for mouse serotonin 5-HT3 receptor and rat and human purinoreceptor P2x subtypes were introduced into the pSFV1 vector. In vitro transcribed RNAs were coelectroporated with pSFV-Helper2 RNA into BHK cells, where in vivo packaging resulted in high titer SFV-5-HT3 and SFV-P2x virus stocks. Infection of BHK, CHO and RJN cells resulted in high-level expression of recombinant receptors. Saturation binding analysis indicated the presence of more than 3 × 106 5-HT3 receptors per cell. Binding studies on isolated membranes yielded from 10 to 60 pmol of either 5-HT3 or P2x receptor per mg protein. Functional responses to the P2x receptors were demonstrated in SFV-infected CHO cells by Ca2+ mobilization or by 45Ca2+ influx. High amplitude electrophysiological responses were also detected for both SFV-5-HT3 and SFV-P2x infected CHO cells in whole-cell patch clamp recordings. To facilitate the purification procedure of SFV-expressed recombinant receptors a histidine tag was introduced at the C-terminus of the 5-HT3 receptor. This 5-HT3His receptor showed high levels of expression, specific binding and high amplitude electrophysiological responses. For large scale expression the BHK cells were adapted to suspension culture and were efficiently infected in a 11.5 liter fermentor culture with SFV-5-HT3His resulting in high-level expression, 52 pmol receptor per mg protein corresponding to 3.2 × 106 receptors per cell. 相似文献
14.
Effects of temperature, and availability of nitrogen and phosphorus on the abundance of Anabaena and Microcystis in Lake Biwa, Japan: an experimental approach 总被引:23,自引:0,他引:23
Under optimal nutrient conditions, both Microcystis sp. and Anabaena sp. isolated from Lake Biwa grew optimally at 28–32°C but differed in maximal growth rates, phosphate uptake kinetics, maximal
phosphorus quotas, and growth responses to nitrogen and phosphorus limitation. The maximal growth rates of Microcystis and Anabaena were 1.6 and 1.25 divisions day−1, respectively. With phosphate and nitrate in the growth-limiting range, the growth of Microcystis was optimal at an N : P ratio of 100 : 1 (by weight) and declined at lower (nitrogen limitation) and higher (phosphorus limitation)
ratios. In contrast, Anabaena growth rates did not change at N : P ratios from 1000 : 1 to 10 : 1. Starting with cells containing the maximal phosphorus
quota, Microcystis growth in minus-phosphorus medium ceased in 7–9 days, compared with 12–13 days for Anabaena. The phosphate turnover time in cultures starved to their minimum cell quotas was 7.9 min for Microcystis and 0.6 min for Anabaena. Microcystis had a higher K
s (0.12 μg P l−1 10−6 cells) and lower V
max (9.63 μg P l−1 h−1 10−6 cells), than Anabaena (K
s 0.02 μg P l−1 h−1 10−6 cells; V
max 46.25 63 μg P l−1 h−1 10−6 cells), suggesting that Microcystis would not be able to grow well in phosphorus-limited waters. We conclude that in spite of the higher growth rate under ideal
conditions, Microcystis does not usually bloom in the North Basin because of low availability of phosphorus and nitrogen. Although Anabaena has an efficient phosphorus-uptake system, its main strategy for growth in low-phosphorus environments may depend on storage
of phosphorus during periods of abundant phosphorus supply, which are rare in the North Basin.
Received: July 31, 2000 / Accepted: October 18, 2000 相似文献
15.
A novel endo-type β-agarase gene, agaA, was cloned from a newly isolated marine bacterium, Agarivorans sp. LQ48. It encodes a protein of 457 amino acids with a calculated molecular mass of 51.2 kDa. The deduced protein contains
a typical N-terminal signal peptide of 25 amino acid residues, followed by a catalytic module, which is homologous to that
of glycoside hydrolase family 16. A sequence similar to a carbohydrate-binding module is found in the C-terminal region of
the enzyme. The overall amino acid sequence shares a highest identity of 73% with the sequence of beta-agarase AgaB from Pseudoalteromonas sp. strain CY24. The mature agarase was highly expressed extracellularly in Escherichia coli. At pH 7.0 and 40°C, the purified recombinant AgaA had a high specific activity of 349.3 μmol min−1 mg−1, a K
m of 3.9 mg ml−1, and a V
max of 909.1 μmol min−1 mg−1 for agarose. The recombinant enzyme hydrolyzed the β-1,4-glycosidic linkages of agarose, yielding neoagarotetraose and neoagarohexaose
as the main products. Enzyme activity analysis revealed that the optimal temperature and pH of the recombinant AgaA were 40°C
and 7.0, respectively. Notably, AgaA still retained more than 95% activity after incubation at pH 3.0–11.0 for 1 h, a characteristic
much different from other agarases reported. It is the first agarase identified to have so wide a pH range stability. This
favorable property could make AgaA to be attractive to the food, cosmetic, and medical industrial applications. 相似文献
16.
Carinhas N Bernal V Yokomizo AY Carrondo MJ Oliveira R Alves PM 《Applied microbiology and biotechnology》2009,81(6):1041-1049
One of the major concerns regarding the use of insect cells and baculovirus expression vectors for the production of recombinant
proteins is the drop in production observed when infecting cultures at high cell densities; this work attempts to understand
this so-called cell density effect in the scope of baculovirus production for gene therapy purposes. A Spodoptera frugiperda insect cell line (Sf-9) was cultured and infected in serum-free medium, and the patterns of production of a recombinant baculovirus
expressing the green fluorescent protein (GFP) were analyzed at different cell concentrations at infection (CCIs) and multiplicities
of infection (MOIs). The results confirm that a cell density effect on productivity occurs which is dependent on the MOI used,
with a high MOI “delaying” the drop in production to higher cell densities. Medium replacement at the time of infection using
a high MOI considerably improved baculovirus production, with the different production indicators, namely the titer, specific
yield, amplification factor, and time of harvesting, increasing with cell concentration for the CCI range tested. Virus titers
as high as 2.6 × 1010 IP.mL−1 were obtained in cultures infected at 3.5 × 106 cells.mL−1, while the amplification factor was roughly 19 times higher than the highest value obtained without medium exchange. 相似文献
17.
Different nutrient-feeding cultures were carried out in producing recombinant protein of truncated tumor necrosis factor related apoptosis-inducing ligand (TRAIL) (114–281 amino acids of TRAIL) in Escherichia coli strain C600/pBV-TRAIL. The effects of preinduction specific growth rate, postinduction carbon source (glucose and glycerol), and feeding strategies were investigated. The higher preinduction specific growth rate (μ=0.22 h−1) contributed to the increase in the TRAIL production, at which TRAIL was accumulated in bacterial cells as 7.2% of total cellular protein, corresponding to 1.99 g l−1 in contrast with 5.1% (1.29 g l−1) at preinduction specific growth rate (μ=0.1 h−1) during high-cell-density culture. Glycerol was superior to glucose as the postinduction carbon source for TRAIL production. Under similar culture conditions, the final concentration of TRAIL was produced 1.59-fold more when glycerol was used as postinduction carbon source than when glucose was used. At the same time, the results showed that it is efficient to adopt the pH-stat feeding strategy at postinduction for the overproduction of TRAIL. The TRAIL production was increased up to 4.51 g l−1, approximately 16.1% of total cellular protein. The mechanisms behind the preinduction specific growth rate effect on the expression level may be ascribed to the leakage secretion of acetate. 相似文献
18.
The endochitinase DNA and cDNA from Trichoderma sp. were cloned, sequenced and expressed. The cloned DNA and cDNA sequences were 1,476 and 1,275 bp in length, respectively.
There were three introns in DNA sequence in comparison with the cDNA sequence. The endochitinase protein contained three regions:
the signal peptide, the prepro-region and the mature protein region. The gene fragment encoding the mature endochitinase was
ligated into the expression vector pET-28a+, yielding pET-1. The plasmid pET-1 was transformed into the Escherichia coli BL21 (DE3). The clone bearing pET-1 was picked and cultured at 30°C for the expression of endochitinase. SDS-PAGE analysis
showed that the endochitinase was expressed in the periplasmic space and the purified protein showed a single band. The activity
of 70.2 U/mg was obtained from the cellular extract of the recombinant strain. The activity of endochitinase was 2.5-fold
higher at 24 h than at 16 h in the periplasmic space. The optimal pH and temperature of the recombinant endochitinase were
determined to be 7.0 and 35°C, respectively. It was relatively stable within the pH range of 5–8. Significant activity stimulation
by 1 mM Mg2+ and 5 mM Fe2+ and inhibition by 5 mM Co2+ and 5 mM Hg2+ were observed. The kinetic constants Km, Vmax and Kcat for the hydrolysis of the colloidal chitin were 1.5 mM, 1.37 μmol min−1 and 6.23 min−1, respectively. 相似文献
19.
The effects of three organic compounds were tested on one of the most used marine micro-algae in the aquaculture of molluscs
and crustaceans, Tetraselmis suecica. Studies were made in axenic conditions with yeast extract, peptone and glucose added to the culture medium, each alone,
in combinations of two or all together. Medium without any organic compound was used for the control. Cultures containing
yeast extract grew best, reaching maximum cell density of 3.79 × 106 and 3.84 × 106 cells ml−1.
The organic carbon source affected the biochemical composition. The components most affected were the carbohydrates, with
values between 6.5 pg cell−1 in control cultures and 48.5 pg cell−1 in glucose cultures. Protein content ranged between 27.5 pg cell−1 in control cultures and 88.6 pg cell−1 in yeast + glucose + peptone cultures. The lipid content changed little. Maximum protein yields were reached in cultures
with yeast + glucose and with yeast - glucose - peptone, with values of 24.6 and 28.2 mg 1−1 d−1, respectively. These values are 22 and 25 times those in control cultures. A maximum carbohydrate yield of 7.9 mg carbohydrate
per litre per day was obtained in yeast + glucose + peptone cultures, 27 times that in the control cultures. The maximum lipid
yield was obtained with yeast + glucose + peptone and yeast + glucose. Maximum energy values were 308 kcal 1− in yeast extract - glucose - peptone cultures and 279 kcal 1−1 in yeast extract + glucose cultures. Gross energy values in control cultures were 24.5 kcal 1−1, but peptone cultures presented the minimum energy value, 22 kcal 1−1. The yeast extract: glucose ratio in the culture medium was optimized. A ratio 2:1 produced the best yields in cells, protein,
carbohydrate and gross energy. 相似文献
20.
Extracellular human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression was studied under the control of
the GAP promoter in recombinant Pichia pastoris in a series of continuous culture runs (dilution rates from 0.025 to 0.2 h−1). The inlet feed concentration was also varied and the steady state biomass concentration increased proportionally demonstrating
efficient substrate utilization and constancy of the biomass yield coefficient (Yx/s) for a given dilution rate. The specific product formation rate (qP) showed a strong correlation with dilution rates demonstrating growth associated product formation of hGM-CSF. The volumetric
product concentration achieved at the highest feed concentration (4×) and a dilution rate of 0.2 h−1 was 82 mg l−1 which was 5-fold higher compared to the continuous culture run with 1× feed concentration at the lowest dilution rate thus
translating to a 40 fold increase in the volumetric productivity. The specific product yield (YP/X) increased slightly from 2 to 2.5 mg g−1, with increasing dilution rates, while it remained fairly invariant, for all feed concentrations demonstrating negligible
product degradation or feed back inhibition. The robust nature of this expression system would make it easily amenable to
scale up for industrial production. 相似文献