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1.
We have developed Cucumber mosaic virus (CMV) as a plant virus vector especially for production of pharmaceutical proteins. The CMV vector is a vector modifiable
for different host plants and does not require further engineering steps. CMV contains three genomic RNA molecules (RNAs 1–3)
necessary for infectivity. With this system, instead of creating different vector constructs for each plant we use, we take
advantage of the formation of pseudrecombinants between two CMV isolates by simply reassembling a vector construct (RNA 2
base) and an RNA molecule containing the host determinant (mostly RNA 3). In this study, the gene for acidic fibroblast growth
factor (aFGF), one of the human cytokines, was cloned under the control of the subgenomic promoter for RNA 4A of the CMV-based
vector, C2-H1. Infected Nicotiana benthamiana plants produced aFGF at levels up to 5–8% of the total soluble protein. The tobacco-produced aFGF was purified, and its biological
activity was confirmed. Using this system, which provides a versatile and viable strategy for the production of therapeutic
proteins in plants, we also demonstrated a high level of aFGF in Glycine max (soybean) and Arabidopsis thaliana. 相似文献
2.
Shahin Emami Wilma Merrill Van Cherington Gisela G. Chiang Michael Kirchgesser Joseph M. Appel Michael Hansen Peter H. Levine Joel S. Greenberger David R. Hurwitz 《In vitro cellular & developmental biology. Animal》1997,33(7):503-511
Summary The ex vivo establishment, expansion, transduction, and reintroduction of autologous bone marrow stromal cells offers a potential efficacious
system for somatic cell gene therapy. It is likely that any ex vivo system will require the use of large numbers of cells which express high levels of transgene products. We present a method
for routine expansion of canine bone marrow stromal cells, established from initial 10–20 ml marrow aspirates, to greater
than 109 cells. This high level expansion of cell cultures uses the stimulatory effect of acidic fibroblast growth factor (aFGF) and
heparin. In the absence of these factors, stromal cell cultures grow actively for only 1 to 2 passages, become flattened in
morphology, and expand to only 108 cells. In the presence of heparin (5 U/ml), aFGF exerts its effect over a wide range of concentrations (0.1–10 ng/ml) in
a dose-dependent manner. The stimulatory effect is dependent on the presence of both aFGF and heparin. Immunocytochemical
and cytochemical analyses phenotypically characterize these stromal cells as bone marrow stromal myofibroblasts. Stromal cells
grown in the presence of aFGF and heparin grow actively and maintain a fibroblast-like morphology for a number of passages,
transduce efficiently with a human growth hormone (hGH) expression vector, and express and secrete high levels of hGH. Human
marrow stromal cells were also established and expanded by the same culture method. This culture method should be of great
value in somatic cell gene therapy for the delivery of secreted gene products to the plasma of large mammals. 相似文献
3.
α-Agarase hydrolyzes the α-1,3 linkage of agarose yielding agaro-oligosaccharides. It is less well characterized than β-agarase.
AgaA gene (2.3 kb ORF), encoding the α-agarase from Thalassomonas JAMB A33, was subcloned into both a constitutive and an inducible expression vector. Both the constructed plasmids, pVT-AgaA
(ADH1 promoter) and pYInu-AgaA (GAL10 promoter), were transformed into Saccharomyces cerevisiae SEY2102 and FY833 and pPIC9-AgaA harboring the AOX1 promoter was transformed into Pichia pastoris GS115. The recombinant α-agarases were over-expressed with activities from 0.3 to 1.6 unit/ml, the highest being in the SEY2102/pYInu-AgaA
transformant. Most of the recombinant α-agarase was extracellular because each plasmid possesses a signal sequence for the
secretory production of α-agarase. In contrast, the Pichia host-vector expression system was unsuitable for the production of recombinant α-agarase. This is the first report of recombinant
production of α-agarase in yeast for industrial use. 相似文献
4.
Jeong Seok Oh Hee Ho Park Tai Hyun Park 《Biotechnology and Bioprocess Engineering》2008,13(4):470-475
In a two-phase operation, E. coli containing λSNNU1 (Q
−
S
−) in the chromosome is typically cultured at 33°C and cloned gene expression is induced by elevating the temperature. At least
40°C is necessary for complete induction of cloned gene expression; however, temperatures above 40°C have been shown to inhibit
cloned gene expression. This suggests that a three-phase operation, which has an induction phase between the growth and production
phases, may result in higher gene expression. In this study, optimal temperature management strategies were investigated for
the three-phase operation of cloned gene expression in thermally inducible E. coli/bacteriophage systems. The optimal temperature for the induction phase was determined to be 40°C. When the temperature of
the production stage was 33°C, the optimal time period for the induction phase at 40°C was determined to be 60 min. In contrast,
when the temperature of the production phase was 37°C, the optimal period for the induction phase at 40°C was 20∼30 min. When
the three-phase temperature and temporal profile were set at a growth phase of 33°C, an induction phase at 40°C for 30 min,
and a production phase at 37°C, the highest level of cloned gene expression was achieved. 相似文献
5.
Noushin Davoudi Azam Hemmati Zahra Khodayari Ahmad Adeli Mahdi Hemayatkar 《World journal of microbiology & biotechnology》2011,27(8):1893-1899
Increasing therapeutic applications for recombinant human interferon-gamma (rhIFN-γ), an antiviral pro-inflammatory cytokine,
has broadened interest in optimizing methods for its production. We herein describe a unicellular eukaryotic system, Leishmania tarentolae, a Trypanosomatidae protozoan parasite of gecko Tarentola annularis, which has recently been introduced as a candidate for heterologous gene expression. In this study, the hIFN-γ cDNA was amplified from phyto-hemagglutinin-stimulated peripheral blood mononuclear cells of a healthy blood donor using
RT–PCR. In order to express, the rhIFN-γ protein, the resulting cDNA was cloned in two expression cassettes (each containing
one copy of hIFN-γ cDNA) and integrated into the small subunit of ribosomal RNA gene of L. tarentolae genome by electroporation. Transformed clones were selected in the presence of appropriate antibiotics. Western blotting
of rhIFN-γ and ELISA confirmed the expression and production of 9.5 mg of rhIFN-γ protein/l respectively. 相似文献
6.
Y. Shiba F. Fukui K. Ichikawa N. Serizawa H. Yoshikawa 《Applied microbiology and biotechnology》1998,50(1):34-41
In order to develop a production process for carboxypeptidase Y (CPY, yeast vacuolar protease) secreted by Saccharomyces cerevisiae KS58-2D, medium composition, culture conditions, and expression systems were investigated. We found that the addition of
histidine to thiamine-free medium, in which CPY production was almost negligible, raised the intracellular thiamine level,
resulting in the increase of CPY production. On the basis of the choice of an expression system that uses an inducible GAL10 promoter, reassessment of histidine concentration in the medium, and optimization of the pH level during cultivation (pH
6.5), active CPY was secreted in a quantity of over 400 mg/l, which was more than tenfold that higher than that previously
reported. The process developed could be easily scaled-up to industrial-scale fermentation.
Received: 16 January 1998 / Received revision: 16 February 1998 / Accepted: 27 February 1998 相似文献
7.
High-level expression of a recombinant protein in Klebsiella planticola owing to induced secretion into the culture medium 总被引:1,自引:0,他引:1
G. Miksch R. Neitzel K. Friehs E. Flaschel 《Applied microbiology and biotechnology》1999,51(5):627-632
The Tn5-based transposon Tn5-KIL3 (Miksch et al. 1997c) bearing the kil gene of the ColE1 plasmid of Escherichia coli, which mediates controlled export of periplasmic proteins into the culture medium, was stably integrated into the chromosome
of Klebsiella planticola with high transposition frequency. A Bacillus hybrid β-glucanase located on an RSF1010-derived plasmid was mobilized from E.coli to K. planticola and used as a reporter protein to select strains with high expression and secretion competence. During fermentation experiments
it was shown that the production of β-glucanase in K. planticola was improved to an unexpectedly high level when the enzyme was secreted into the medium. Due to the stationary-phase promoter
used for the expression of the kil gene the secretion of β-glucanase into the medium started at the transition from the exponential to the stationary phase,
as in E. coli, and the fraction of secreted protein reached 90%. The results showed that K. planticola may represent an interesting organism for the production of heterologous proteins.
Received: 22 July 1998 / Received revision: 25 November 1998 / Accepted: 29 November 1998 相似文献
8.
9.
10.
Strains of the fission yeast Schizosaccharomyces pombe have been constructed containing single or multiple chromosomally integrated copies of an expression cassette for production
of human gastric lipase. Integrant strains of S. pombe secrete active lipase and are stable for lipase production over a minimum of 50 generations in non-selective media. Lipase
activity levels for integrant strains containing up to three tandem copies of the expression cassette are strongly correlated
with copy number of the cassette in both complete and minimal media. Lipase activity is higher in complete medium than in
minimal medium. Strains carrying three chromosomally integrated expression cassette copies can be grown without selection
in complete medium and are capable of significantly higher lipase activities than strains containing the expression cassette
on a multicopy plasmid.
Received: 27 March 1997 / Received revision: 13 August 1997 / Accepted: 25 August 1997 相似文献
11.
Díaz-Barrera A Soto E Altamirano C 《Journal of industrial microbiology & biotechnology》2012,39(4):613-621
Alginates are polysaccharides that are used as thickening agents, stabilizers, and emulsifiers in various industries. These
biopolymers are produced by fermentation with a limited understanding of the processes occurring at the cellular level. The
objective of this study was to evaluate the effects of agitation rate and inlet sucrose concentrations (ISC) on alginate production
and the expression of the genes encoding for alginate-lyases (algL) and the catalytic subunit of the alginate polymerase complex (alg8) in chemostat cultures of Azotobacter vinelandii ATCC 9046. Increased alginate production (2.4 g l−1) and a higher specific alginate production rate (0.1 g g−1 h−1) were obtained at an ISC of 15 g l−1. Carbon recovery of about 100% was obtained at an ISC of 10 g l−1, whereas it was close to 50% at higher ISCs, suggesting that cells growing at lower sucrose feed rates utilize the carbon
source more efficiently. In each of the steady states evaluated, an increase in algL gene expression was not related to a decrease in alginate molecular weight, whereas an increase in the molecular weight of
alginate was linked to higher alg8 gene expression, demonstrating a relationship between the alg8 gene and alginate polymerization in A. vinelandii for the first time. The results obtained provide a possible explanation for changes observed in the molecular weight of alginate
synthesized and this knowledge can be used to build a recombinant strain able to overexpress alg8 in order to produce alginates with higher molecular weights. 相似文献
12.
Human kringle domains (KDs) are ubiquitously expressed binding modulators that fold into seven flexible loops and it has been
previously demonstrated that KDs can be engineered toward target-specific binding proteins as a non-antibody protein scaffold.
Here, we report a method for efficient expression of a KD derivative (KD548)—a promising anti-cancer agent—by high-cell-density
culture of Escherichia coli at a preparative scale production. The correct folding of KD548 requires three disulfide bonds. Nevertheless, cytoplasmic
expression of KD548 in E. coli led to good yields of highly soluble proteins with high activity. For efficient expression, four sets of expression systems
consisting of different promoters (lac or T7) and fusion tags (His or FLAG) were examined. Of these, the expression system using a combination of the T7 promoter
with the FLAG tag resulted in the highest production in shake flask cultivation as well as in high-cell-density cultivation
performed in a 6.6-L jar bioreactor. When protein expression was induced at high-cell density (optical density [OD] = 100)
and when complex feeding solutions were supplemented, cell density (maximum OD = 184) and production yield (∼5.4 g/L) were
significantly enhanced to values that were much higher than those found previously with Pichia cultivation (<8 mg/L). 相似文献
13.
14.
An expression vector constructed from genes of Pichia pastoris was applied for heterologous gene expression in Saccharomyces cerevisiae. Recombinant hepatitis B surface antigen was synthesized by cloning hepatitis B virus ‘S’ gene under the control of glyceraldehyde-3-phosphate
dehydrogenase (GAP) promoter of Pichia pastoris in Saccharomyces cerevisiae. Hepatitis B surface antigen was constitutively expressed, was stable and exhibited ∼20–22 nm particle formation. Stability
and absence of toxicity to the host with the expression vector indicates the expression system can be applied for large-scale
production. 相似文献
15.
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
16.
D H Lee K Uchiyama S Shioya Y I Hwang 《Journal of industrial microbiology & biotechnology》1998,20(3-4):160-165
In order to maximize the glucoamylase production by recombinant Saccharomyces cerevisiae in batch culture, first a temperature-controlled expression system for a foreign gene in S. cerevisiae was constructed. A temperature-sensitive pho80 mutant of S. cerevisiae for the PHO regulatory system, YKU131, was used for this purpose. A DNA fragment bearing the promoter of the PHO84 gene, which encodes an inorganic phosphate (Pi) transporter of S. cerevisiae and is derepressed by Pi starvation, was used as promoter. The glucoamylase gene connected with the PHO84 promoter was ligated into a YEp13 vector, designated pKU122. When the temperature-sensitive pho80
ts mutant harboring the plasmid pKU122 is cultivated at a lower temperature, the expression of glucoamylase gene is repressed,
but at a higher temperature it is expressed. Next the effect of temperature on the specific growth rate, μ, and specific production
rate, ρ, was investigated. Maximum values of ρ and ρ at various temperatures were at 30°C and 34°C, respectively. The optimal
cultivation temperature strategy for maximum production of glucoamylase by this recombinant strain in batch culture was then
determined by the Maximum principle using the relationships of μ and ρ to the cultivation temperature. Finally, the optimal
strategy was experimentally realized by changing the cultivation temperature from Tμ (30°C) to Tρ (34°C) at the switching
time, ts.
Received 18 September 1997/ Accepted in revised form 07 January 1998 相似文献
17.
Shu-Ming Wu Chi Feng Jin Zhong Lian-Dong Huan 《World journal of microbiology & biotechnology》2011,27(1):99-106
Nattokinase (NK) is a health product for the prevention and potential control of thrombosis diseases. To explore the possibility
of enhancing NK production in Bacillus subtilis by altering the promoter of NK gene (PaprN), we tested several methods. We substituted the wild-type −10 box (TACAAT) of PaprN with the consensus sequence (TATAAT) of σA-dependent promoters, mutated the original −35 box (TACTAA) to a partial consensus sequence (TACACA), and expressed aprN from two tandem promoters, respectively. The efficacies of these changes were monitored by fibrinolytic activity, SDS-PAGE,
and northern blotting analyses. Fibrinolytic activity analysis showed that altering the −10 region of PaprN could increase NK production by 136%. This production is significantly higher than those reported in the literatures. Similar
results were obtained in SDS-PAGE and northern blotting analyses. This engineered promoter was also able to enhance the expression
of β-glucuronidase (GUS) by 249%. Partial alteration of the −35 element could slightly improve the production of NK by 13%,
while two tandem promoters just had marginal effects on the production of NK. Our study showed that alteration of −10 or −35
elements in PaprN, especially −10 element, is an effective way to enhance the production of heterologous proteins in B. subtilis. 相似文献
18.
CCN2 (Connective Tissue Growth Factor) is essential for extracellular matrix production and integrin signaling in chondrocytes 总被引:2,自引:0,他引:2
Nishida T Kawaki H Baxter RM Deyoung RA Takigawa M Lyons KM 《Journal of cell communication and signaling》2007,1(1):45-58
The matricellular protein CCN2 (Connective Tissue Growth Factor; CTGF) is an essential mediator of ECM composition, as revealed through analysis of Ccn2 deficient mice. These die at birth due to complications arising from impaired endochondral ossification. However, the mechanism(s)
by which CCN2 mediates its effects in cartilage are unclear. We investigated these mechanisms using Ccn2
−/−
chondrocytes. Expression of type II collagen and aggrecan were decreased in Ccn2
−/− chondrocytes, confirming a defect in ECM production. Ccn2
−/−
chondrocytes also exhibited impaired DNA synthesis and reduced adhesion to fibronectin. This latter defect is associated
with decreased expression of α5 integrin. Moreover, CCN2 can bind to integrin α5β1 in chondrocytes and can stimulate increased
expression of integrin α5. Consistent with an essential role for CCN2 as a ligand for integrins, immunofluorescence and Western
blot analysis revealed that levels of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK)1/2 phosphorylation
were reduced in Ccn2
−/−
chondrocytes. These findings argue that CCN2 exerts major effects in chondrocytes through its ability to (1) regulate ECM
production and integrin α5 expression, (2) engage integrins and (3) activate integrin-mediated signaling pathways. 相似文献
19.
Sygmund C Gutmann A Krondorfer I Kujawa M Glieder A Pscheidt B Haltrich D Peterbauer C Kittl R 《Applied microbiology and biotechnology》2012,94(3):695-704
Pyranose dehydrogenase (PDH) is a fungal flavin-dependent sugar oxidoreductase that is highly interesting for applications
in organic synthesis or electrochemistry. The low expression levels of the filamentous fungus Agaricus meleagris as well as the demand for engineered PDH make heterologous expression necessary. Recently, Aspergillus species were described to efficiently secrete recombinant PDH. Here, we evaluate recombinant protein production with expression
hosts more suitable for genetic engineering. Expression in Escherichia coli resulted in no soluble or active PDH. Heterologous expression in the methylotrophic yeast Pichia pastoris was investigated using two different signal sequences as well as a codon-optimized sequence. A 96-well plate activity screening
for transformants of all constructs was established and the best expressing clone was used for large-scale production in 50-L
scale, which gave a volumetric yield of 223 mg L−1 PDH or 1,330 U L−1 d−1 in space–time yield. Purification yielded 13.4 g of pure enzyme representing 95.8% of the initial activity. The hyperglycosylated
recombinant enzyme had a 20% lower specific activity than the native enzyme; however, the kinetic properties were essentially
identical. This study demonstrates the successful expression of PDH in the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, the feasibility of large-scale production of the enzyme with this expression
system together with a simplified purification scheme for easy high-yield purification is shown. 相似文献
20.
Böer E Breuer FS Weniger M Denter S Piontek M Kunze G 《Applied microbiology and biotechnology》2011,92(1):105-114
Tannase (tannin acyl hydrolase, EC 3.1.1.20) hydrolyses the ester and depside bonds of gallotannins and gallic acid esters
and is an important industrial enzyme. In the present study, transgenic Arxula adeninivorans strains were optimised for tannase production. Various plasmids carrying one or two expression modules for constitutive expression
of tannase were constructed. Transformant strains that overexpress the ATAN1 gene from the strong A. adeninivorans TEF1 promoter produce levels of up to 1,642 U L−1 when grown in glucose medium in shake flasks. The effect of fed-batch fermentation on tannase productivity was then investigated
in detail. Under these conditions, a transgenic strain containing one ATAN1 expression module produced 51,900 U of tannase activity per litre after 142 h of fermentation at a dry cell weight of 162 g L−1. The highest yield obtained from a transgenic strain with two ATAN1 expression modules was 31,300 U after 232 h at a dry cell weight of 104 g L−1. Interestingly, the maximum achieved yield coefficients [Y(P/X)] for the two strains were essentially identical. 相似文献