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  • Articles  (14)
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  • gene expression
  • Springer  (14)
  • 1995-1999  (9)
  • 1990-1994  (5)
  • Process Engineering, Biotechnology, Nutrition Technology  (14)
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  • Articles  (14)
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  • 1
    Electronic Resource
    Electronic Resource
    Quantitative analysis of transcription and translation in gene amplified Chinese hamster ovary cells on the basis of a kinetic model (1999)
    Springer
    Cytotechnology 29 (1999), S. 93-102 
    Details
    ISSN: 1573-0778
    Keywords: CHO cells ; gene expression ; kinetic model ; protein secretion ; transcription ; translation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The elevation of expression levels for secreted glycoproteins by gene amplification in mammalian cells shows a saturation behavior at high levels of gene amplification. At high expression levels a drop in the secretion efficiency for the recombinant protein occurs (Schröder and Friedl, 1997), coinciding with the appearance of misfolded protein in the cell. In this communication we investigated whether additional limitations exist at the levels of transcription and translation. Four Chinese hamster ovary (CHO) cell lines expressing different amounts of human antithrombin III (ATIII) were used as a model system. A tenfold increase in the ATIII cDNA copy number from the lowest to the highest producing cell line coincided with a 38-fold increase in ATIII mRNA levels, and an 80-fold increase in the amount of intracellular ATIII levels. The data was analyzed using a simple kinetic model. The following conclusions were derived: I. The transcriptional activity for the recombinant protein is not saturated. II. Translation itself is not saturated either, but may be downregulated as secretion efficiency drops. III. Two explanations for the previously reported drop in secretion efficiency for the recombinant protein with increasing expression level are possible: A. Protein degradation is an alternative fate for translated ATIII and the fraction of ATIII degraded after translation increases as expression level is increased. B. Translation is downregulated as the secretory apparatus becomes exhausted to maintain cell viability.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008077603328
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  • 2
    Electronic Resource
    Electronic Resource
    Embryonic stem cell differentiation models: cardiogenesis, myogenesis, neurogenesis, epithelial and vascular smooth muscle cell differentiation in vitro (1999)
    Springer
    Cytotechnology 30 (1999), S. 211-226 
    Details
    ISSN: 1573-0778
    Keywords: cardiogenesis ; cell differentiation ; gene expression ; mouse embryonic stem cells ; myogenesis ; neurogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent cell lines of undifferentiated cells. ES cells provide an important cellular system in developmental biology for the manipulation of preselected genes in mice by using the gene targeting technology. Embryonic stem cells, when cultivated as embryo-like aggregates, so-called ‘embryoid bodies’, are able to differentiate in vitro into derivatives of all three primary germ layers, the endoderm, ectoderm and mesoderm. We established differentiation protocols for the in vitro development of undifferentiated embryonic stem cells into differentiated cardiomyocytes, skeletal muscle, neuronal, epithelial and vascular smooth muscle cells. During differentiation, tissue-specific genes, proteins, ion channels, receptors and action potentials were expressed in a developmentally controlled pattern. This pattern closely recapitulates the developmental pattern during embryogenesis in the living organism. In vitro, the controlled developmental pattern was found to be influenced by differentiation and growth factor molecules or by xenobiotics. Furthermore, the differentiation system has been used for genetic analyses by ‘gain of function’ and ‘loss of function’ approaches in vitro.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008041420166
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  • 3
    Electronic Resource
    Electronic Resource
    Transient gene expression in mammalian cells grown in serum-free suspension culture (1999)
    Springer
    Cytotechnology 30 (1999), S. 71-83 
    Details
    ISSN: 1573-0778
    Keywords: gene expression ; HEK293(EBNA) cells ; serum-free ; transient transfection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract In order to establish a simple and scaleable transfection system we have used the cationic polymer polyethylenimine (PEI) to study transient transfection in HEK293 and 293(EBNA) cells grown in serum-free suspension culture. The transfection complexes were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI transfection complexes were prepared in fresh medium (1/10 culture volume) and then added to the cells (indirect method). The results of this study clearly show that the ratio of PEI nitrogen to DNA phosphate is very important for high expression levels. The precise ratio is dependent on the DNA concentration. For example, using 1 μg/ml DNA by the indirect method, the ratio of optimal PEI:DNA was about 10–13:1. However, the ratio increases to 33:1 for 0.1–0.2 μg/ml DNA. By testing several different molecular weights of the polycationic polymer we could show that the highest transfection efficiency was obtained with the PEI 25 kDa. Using PEI 25 kDa the indirect method is superior to the direct addition because significantly lower DNA concentrations are needed. The expression levels of the soluble human TNF receptor p55 are even higher at low DNA compared to 1 μg/ml plasmid. The EBV-based pREP vectors gave better transient gene expression when used in 293(EBNA) cells compared to HEK293 cells in suspension culture. No differences in expression levels in the two cell lines were observed when the pC1 (CMV)-TNFR was used. In conclusion, PEI is a low-toxic transfection agent which provides high levels of transient gene expression in 293(EBNA) cells grown in serum-free suspension culture. This system allows highly reproducible, cost-effective production of milligram amounts of recombinant proteins in 2–5 l spinner culture scale within 3–5 days. Fermentor scale experiments, however, are less efficient because the PEI-mediated transient tranfection is inhibited by conditioned medium.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008000327766
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  • 4
    Electronic Resource
    Electronic Resource
    Stable production of an analog of human tissue plasminogen activator from culturedDrosophila cells (1992)
    Springer
    Cytotechnology 10 (1992), S. 157-167 
    Details
    ISSN: 1573-0778
    Keywords: Drosophila cell culture ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We have studied the expression of an analog of human tissue plasminogen activator, FK2P, inDrosophila Schneider 2 cells. A number of promoters were tested, including theDrosophila metallothionein promoter (MTd), baculovirus immediate early promoter (IE),Drosophila copia promoter, mouse metallothionein promoter, cytomegalovirus immediate early promoter with or without intron, SV40 immediate early promoter, and human elongation factor 1α promoter. Two of these promoters drove significant expression of FK2P. The MTd promoter is tightly regulated and upon induction with copper or cadmium expression of FK2P increases as much as 180-fold, accumulating in the culture medium to about 7 μg FK2P/106 cells/day as determined by ELISA. The IE promoter can direct the constitutive expression to yield about 0.4 μg FK2P/106 cells/day. The production of FK2P in these cell lines remains at about the same level after repeated passages, even in the absence of selective pressure. The FK2P accumulated in the culture medium is fully active in an assay using a chromogenic substrate for serine proteases. Western immunoblot analysis shows that the product remains predominately as single-chain molecules in serum-free medium, while in serum-containing medium two-chain material occurs as expected due to the presence of plasmin in serum. Judged from the size in Western immunoblots, the FK2P produced is glycosylated.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00570892
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  • 5
    Electronic Resource
    Electronic Resource
    The role of the cell cycle in determining gene expression and productivity in CHO cells (1999)
    Springer
    Cytotechnology 30 (1999), S. 49-57 
    Details
    ISSN: 1573-0778
    Keywords: cell cycle ; CHO ; flow cytometry ; gene expression ; synchronisation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Understanding the relationships between cell cycle and protein expression is critical to the optimisation of media and environmental conditions for successful commercial operation of animal cell culture processes. Using flow cytometry for the analysis of the early phases of synchronised batch cultures, the dependency of product expression on cell cycle related events has been evaluated in a recombinant CHO cell line. Although the production of recombinant protein is initially found to be cell cycle related, the maximum specific protein productivity is only achieved at a later stage of the exponential phase which also sees a maximum in the intracellular protein concentration. Subsequent work suggests that it is the batch phase/medium composition of cultures which is the major determinant of maximum specific productivity in this cell line. Furthermore the effect of the positive association between S phase and specific productivity is subordinate to the effect of batch phase/medium composition on the specific productivity of batch cultures.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008093404237
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  • 6
    Electronic Resource
    Electronic Resource
    Recombinant protein expression in aDrosophila cell line: comparison with the baculovirus system (1994)
    Springer
    Cytotechnology 15 (1994), S. 139-144 
    Details
    ISSN: 1573-0778
    Keywords: Baculovirus ; cell culture ; Drosophila ; gene expression ; insect cell ; metallothionein promoter ; recombinant protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract In this report, we compare two different expression systems: baculovirus/Sf9 and stable recombinantDrosophila Schneider 2 (S2) cell lines. The construction of a recombinant S2 cell line is simple and quick, and in batch fermentations the cells have a doubling time of 20 hours until reaching a plateau density of 20 million cells/ml. Protein expression is driven by theDrosophila Metallothionein promoter which is tightly regulated. When expressed in S2 cells, the extracellular domain of human VCAM, an adhesion molecule, is indistinguishable from the same protein produced by baculovirus-infected Sf9 cells. Additionally, we present data on the expression of a seven trans-membrane protein, the dopamine D4 receptor, which has been successfully expressed in both systems. The receptor integrates correctly in the S2 membrane, binds [3H]spiperone with high affinity and exhibits pharmacological characteristics identical to that of the receptor expressed in Sf9 and mammalian cells. The general implications for large scale production of recombinant proteins are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00762388
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  • 7
    Electronic Resource
    Electronic Resource
    Molecular biology of extremophiles (1995)
    Springer
    World journal of microbiology and biotechnology 11 (1995), S. 71-84 
    Details
    ISSN: 1573-0972
    Keywords: Archaea ; gene expression ; genetics ; replication ; thermophile
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The molecular biology of extremophiles has recently attracted much interest, both in terms of cell adaptation to extreme environmental conditions and the development of manipulative genetic techniques. Although molecular genetic techniques have been successfully applied to halophiles and methanogens, their use with hyperthermophiles is limited by the extreme growth conditions that these organisms require. Much information on the thermophilic Archaea, has been obtained by studying the key enzymes involved in fundamental cell processes, such as transcription and replication, and by the cloning, sequence comparison and heterologous expression of structural genes. The development of viral vectors and systems for transformation, mutant production and screening will permit increased genetic manipulation of these organisms.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00339137
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  • 8
    Electronic Resource
    Electronic Resource
    Regulation of olfactory neuron gene expression (1993)
    Springer
    Cytotechnology 11 (1993), S. 17-22 
    Details
    ISSN: 1573-0778
    Keywords: olfactory neuroepithelium ; neural development and regeneration ; neuronal phenotype ; gene expression ; OMP ; transgenic mice ; B50/GAP43 ; OLF-1 ; transcription
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00749053
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  • 9
    Electronic Resource
    Electronic Resource
    Isolation, characterization and recombinant protein expression in Veggie-CHO: A serum-free CHO host cell line (1998)
    Springer
    Cytotechnology 28 (1998), S. 31-42 
    Details
    ISSN: 1573-0778
    Keywords: CHO ; DHFR ; gene expression ; growth factor ; recombinant protein ; serum-free
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The dihydrofolate reductase-deficient Chinese hamster ovary cell line, DXB11-CHO, commonly used as a host cell for the production of recombinant proteins requires 7.5% serum-supplementation for optimal growth. Regulatory issues surrounding the use of serum in clinical production processes and the direct and indirect costs of using serum in large-scale production and recovery processes have triggered efforts to derive serum-independent host cell lines. We have successfully isolated a serum-free host that we named Veggie- CHO. Veggie-CHO was generated by adapting DXB11-CHO cells to growth in serum-free media in the absence of exogenous growth factors such as Transferrin and Insulin-like growth factor, which we have previously shown to be essential for growth and viability of DXB11- CHO cells. Veggie-CHO cells have been shown to maintain an average doubling time of 22 hr in continuous growth cultures over a period of three months and have retained the dihydrofolate reductase -deficient phenotype of their parental DXB11-CHO cells. These properties and the stability of its serum-free phenotype have allowed the use of Veggie- CHO as host cells for transfection and amplified expression of recombinant proteins. We describe the derivation a serum-free recombinant cell line with an average doubling time of 20 hr and specific productivity of 2.5 Units recombinant Flt-3L protein per 10e6 cells per day.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008052908496
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  • 10
    Electronic Resource
    Electronic Resource
    Expression of the Aspergillus niger glucose oxidase gene in Penicillium nalgiovense (1995)
    Springer
    World journal of microbiology and biotechnology 11 (1995), S. 322-325 
    Details
    ISSN: 1573-0972
    Keywords: Fungal starter culture ; gene expression ; glucose oxidase ; Penicillium nalgiovense ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The glucose oxidase gene (god) from Aspergillus niger was expressed in Penicillium nalgiovense under control of the latter's homologous transcription signals. The GOD protein was synthesized in an active form, leading to increased glucose oxidase activity. The expression vector was introduced into P. nalgiovense along with a selectable plasmid carrying the dominant amdS marker gene of A. nidulans.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00367109
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  • 11
    Electronic Resource
    Electronic Resource
    Plant genes induced in the Rhizobium-legume symbiosis (1996)
    Springer
    World journal of microbiology and biotechnology 12 (1996), S. 189-202 
    Details
    ISSN: 1573-0972
    Keywords: gene expression ; nodule development ; nodulin genes ; Rhizobium-legume symbiosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Rhizobium, Bradyrhizobium and Azorhizobium can elicit the formation of N2-fixing nodules on the roots or stems of their leguminous host plants. The nodule formation involves several developmental steps determined by different sets of genes from both partners, the gene expression being temporally and spatially coordinated. The plant proteins that are specifically synthesised during the formation and function of the nodule are called nodulins. The nodulins that are expressed before the onset of N2 fixation are termed early nodulins. These proteins are probably involved in the infection process as well as in nodule morphogenesis rather than in nodule function. The nodulins expressed just before or during N2 fixation are termed late nodulins and they participate in the function of the nodule by creating the physiological conditions required for nitrogen fixation, ammonium assimilation and transport. In this review we will describe nodulins, nodulin genes and the relationship between nodulin gene expression and nodule development. The study of nodulin gene expression may provide insight into root-nodule development and the mechanism of communication between bacteria and host plant.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00364683
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  • 12
    Electronic Resource
    Electronic Resource
    Gene expression in Pseudomonas (1993)
    Springer
    World journal of microbiology and biotechnology 9 (1993), S. 433-443 
    Details
    ISSN: 1573-0972
    Keywords: Alginate ; gene expression ; negative regulators ; positive regulators ; promoters ; Pseudomonas ; TOL plasmid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Gene regulation studies in pseudomonad bacteria are mainly restricted to Pseudomonas aeruginosa and Pseudomonas putida. Constitutive promoters exhibit DNA sequences similar to the σ 70-dependent constitutive promoters of Escherichia coli. The TOL meta-cleavage pathway operon promoter and the nah operon promoters are the best characterized σ 70-dependent promoters, which exhibit-10 regions rich in As and Ts and non-conserved-35 regions. The DNA binding motif recognized by the respective positive regulators lies between-40 and-80. Another set of positively controlled promoters exhibit upstream activator sequences located between-100 and-500. Transcription stimulation from some of these promoters also involves σ 54 and/or IHF protein. In this class of promoters, DNA binding is required to establish open complexes. Promoters for the utilization of histidine (hut) are under negative control by the HutC protein. hut promoters exhibit-10/-35 consensus regions and an overlapping operator sequence between-15 and-50. Repression of hut promoters seems to be achieved through steric hindrance of RNA polymerase. Another set of promoters are controlled by catabolite repression, which seems to be cyclic-AMP independent.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00328031
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  • 13
    Electronic Resource
    Electronic Resource
    Expression of fungal genes involved in penicllin biosynthesis (1993)
    Springer
    World journal of microbiology and biotechnology 9 (1993), S. 461-467 
    Details
    ISSN: 1573-0972
    Keywords: Aspergillus nidulands ; catabolite repression ; gene expression ; penicillin ; Penicillium chrysogenum ; Plectomycetes ; regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Carbon catabolite repression and pH regulation are regulatory circuits with a wide domain of action in the Plectomycetes. Penicillin biosynthesis is one of the pathways which are under their control. The conclusions obtained so far, which are based on studies of the genetic and molecular regulation of the penicillin pathway of Aspergillus nidulans, would have been much harder to produce using an organism such as Penicillium chrysogenum (the industrial penicillin producer). However, A. nidulans and P. chrysogenum are close in terms of their phylogeny and one can reasonably predict that the conclusions about A. nidulans, which are summarized in this review and which are of unquestionable biotechnological relevance, will be extrapolable to the industrial organism.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00328034
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  • 14
    Electronic Resource
    Electronic Resource
    Characterization of alginate lyase (ALYII) from Pseudomonas sp. OS-ALG-9 expressed in recombinant Escherichia coli (1999)
    Springer
    World journal of microbiology and biotechnology 15 (1999), S. 105-109 
    Details
    ISSN: 1573-0972
    Keywords: Alginate lyase ; gene expression ; polymannuronate lyase ; Pseudomonas sp.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract An alginate lyase named ALYII was purified to homogeneity from Escherichia coli JM109 carrying a recombinant plasmid, pJK26 harbouring the alyII gene from Pseudomonas sp. OS-ALG-9 by column chromatography with DEAE-cellulose, CM-Sephadex C-50, butyl-Toyopearl 650 M and isoelectric focusing. The molecular size of the purified ALYII was estimated to be 79 kDa by SDS-PAGE and its pI was 8.3. The enzyme was most active at pH 7.0 and 30 °C. Its activity was completely inhibited by Hg2+. The enzyme was poly β-D-1, 4-mannuronate-specific rather than β-D-1, 4-guluronate-specific and it showed a promotion effect in alginate degradation by combination with ALY, an another poly β-D-1, 4-mannuronate-specific alginate lyase from the same strain.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008891100111
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