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  • 1
    Electronic Resource
    Electronic Resource
    Efficient expression of pro-urokinase by human lymphoblastoid Namalwa KJM-1 cells using Moloney retroviral promoter (1995)
    Springer
    Cytotechnology 18 (1995), S. 167-172 
    Details
    ISSN: 1573-0778
    Keywords: efficient gene expression ; Moloney retroviral promoter ; Namalwa KJM-1 ; pro-urokinase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We have compared the level of expression of several enhancer/promoters in human lymphoblastoid Namalwa KJM-1 cells when fused to a common reporter gene. A cassette containing the pro-urokinase (pro-UK) coding sequence followed by the rabbit β-globin and simian virus 40 (SV40) 3′ nontranslated region was used for evaluation of the enhancer activity. Cells containing Moloney murine leukemia virus (Mo-MuLV) promoter had an average of 10–20 fold higher expression levels of pro-UK than those containing other promoters, such as SV40 early gene promoter, human cytomegalovirus (hCMV) major immediate-early gene promoter, Rous sarcoma virus (RSV) promoter and chicken β-actin gene promoter. The expression level of pro-UK under the control of Mo-MuLV promoter was 2–3 μg/106 cells/day and was constant for more than 6 months. Furthermore, the production of a high producer clone, obtained by using dhfr gene coamplification, reached 30–40 μg/106 cells/day. Thus, Mo-MuLV promoter showed the desired characteristics for efficient expression of foreign genes in Namalwa KJM-1 cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00767764
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  • 2
    Electronic Resource
    Electronic Resource
    Stabile production of a thrombin resistant pro-urokinase derivative (PRO-UKS1) by Namalwa KJM-1 cells adapted to serum-free medium (1995)
    Springer
    Cytotechnology 19 (1995), S. 1-10 
    Details
    ISSN: 1573-0778
    Keywords: Namalwa KJM-1 cells ; pro-urokinase ; thrombin resistant ; pro-UKS1 ; stable production ; culture conditions ; serum-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Pro-UKS1 was designed as a thrombin-resistant derivative of pro-urokinase (pro-UK) by introducing a glycosylation site using site-directed mutagenesis. An expression plasmid for pro-UKS1, pMo1UKS1SEd1-5, was constructed and introduced into Namalwa KJM-1 cells (Hosoiet al., 1988), and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UKS1 producer (resistant to 500 nM of MTX), clone 41-8, was selected and further characterized. Clone 41-8 was cultured in serum-free ITPSGF medium (Hosoiet al., 1988). Under the conventional conditions, the concentration of pro-UKS1 reached 26 μg ml−1. Addition of glucose and tri-iodothyronine (T3) improved productivity, and the maximal productivity of pro-UKS1 was 67 μg ml−1 day−1. In this conditioned medium, content of pro-UKS1 was above 80% of total proteins.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00749750
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  • 3
    Electronic Resource
    Electronic Resource
    Expression of human lymphotoxin in Namalwa KJM-1 cells adapted to serum-free medium (1990)
    Springer
    Cytotechnology 4 (1990), S. 39-43 
    Details
    ISSN: 1573-0778
    Keywords: lymphotoxin ; Namalwa KJM-1 ; recombinant DNA technology ; serum-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A Namalwa cell line, KJM-1, which was adapted to serum-free medium is thought to be a good host cell line for recombinant DNA technology. We previously reported the expression of human β-interferon (β-IFN) in Namalwa KJM-1 (Miyaji, 1989a). The utility of Namalwa KJM-1 for expression of foreign genes was further examined. As a target gene to be expressed, human lymphotoxin (hLT) cDNA was used. It was engineered for expression in Namalwa KJM-1 using a simian virus 40 (SV40)-based expression vector pAGE107 (Miyaji, 1989a). It contains all components necessary for the expression of cDNA in mammalian cells. The expression vector was introduced into Namalwa KJM-1 by electroporation. Among the transformants, clone 7 was further examined for the expression of hLT in serum-free medium. The production level of hLT was augmented with the increase of the cell density. Thus it was further indicated that Namalwa KJM-1 is useful for production of foreign gene products.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00148809
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  • 4
    Electronic Resource
    Electronic Resource
    Optimization of cell culture conditions for production of biologically active proteins (1991)
    Springer
    Cytotechnology 5 (1991), S. 17-34 
    Details
    ISSN: 1573-0778
    Keywords: genetic engineering ; Namalwa cells ; perfusion culture ; scaling-up ; serum-free medium ; stable production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We investigated the basic technology of cell culture conditions for production of useful substances such as cytokines, and related proteins produced by Namalwa cells. Namalwa cells (Klein, 1972), human B lymphoblastoid cells, were used for large scale production of alpha-interferon (Klein, 1979). Namalwa KJM-1, a subline of Namalwa cells, adapted to serum- and albumin-free medium, can grow at a high density above 1 × 107 cells/ml in suspension mode by the use of a perfusion culture system, Biofermenter™, containing a cone-type cell-sedimentation column as cell separator (Sato, 1983). Several kinds of cytokine cDNA can be introduced and expressed in Namalwa KJM-1 cells (Miyaji, 1990a,b,c). Some of these were produced in large quantities by use of a gene amplification method with dhfr (Miyaji, 1990c), even though the Namalwa KJM-1 cells contained endogenous dhfr genes. For stable production of the target protein, Namalwa KJM-1 cells are very useful host cells, because they have no effective endogenous protease activity in the conditioned medium. Using Biofermenter with micro-silicone fibers and a dialysis system, the specific productivity of the target proteins was not depressed at a high cell density.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00573878
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  • 5
    Electronic Resource
    Electronic Resource
    Optimization of cell culture conditions for G-CSF (granulocyte-colony stimulating factor) production by genetically engineered namalwa KJM-1 cells (1991)
    Springer
    Cytotechnology 7 (1991), S. 25-32 
    Details
    ISSN: 1573-0778
    Keywords: G-CSF ; high density culture ; Namalwa KJM-1 cells ; optimization ; productivity ; serum-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 μg/ml/day using a 25 cm2 tissue culture flask, even though the cell number was above 7×105 cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, BiofermenterTM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×107 cells/ml), and the amount of G-CSF reached 41 μg/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00135635
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  • 6
    Electronic Resource
    Electronic Resource
    Expression of human beta-interferon in Namalwa KJM-1 which was adapted to serum-free medium (1990)
    Springer
    Cytotechnology 3 (1990), S. 133-140 
    Details
    ISSN: 1573-0778
    Keywords: beta-interferon ; electroporation ; Namalwa KJM-1 ; recombinant DNA technology ; serum-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A Namalwa cell line, KJM-1, which was adapted to serum-free medium is thought to be a very useful host cell line for recombinant DNA technology. Thus, the utility of Namalwa KJM-1 for expression of foreign genes was examined. As a model system human beta-interferon (β-IFN) gene was engineered for expression in this cell line. For construction of the β-IFN expression vector pSE1β1–4, the expression vector pAGE107 was constructed and used. It contains simian virus 40 (SV40) early promoter, the rabbit β-globin RNA processing signals for splicing and polyadenylation, and SV40 early RNA processing signal for polyadenylation. In addition to the above transcription unit, pAGE107 contains the ampicillin-resistance gene and G418-resistance gene. They can confer ampicillin resistance to Escherichia coli (E. coli) and G418 resistance to animal cells. To introduce plasmid DNA into cells, electroporation is a useful method (Wong, 1982; Potter, 1984). We have established conditions for DNA-mediated transfection of Namalwa KJM-1 cell line by electroporation. Among pSE1β1–4-introduced cells, clone 1–3 was further examined for the expression of β-IFN in serum-free medium. The production level of β-IFN was elevated with the increase of the cell density. The results indicated that the Namalwa KJM-1 cell line is useful for production of foreign gene products.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00143675
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  • 7
    Electronic Resource
    Electronic Resource
    High level production of a peptide hormone analogue [Leu13]motilin inE. coli (1988)
    Springer
    Biotechnology letters 10 (1988), S. 763-768 
    Details
    ISSN: 1573-6776
    Source: Springer Online Journal Archives 1860-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary A peptide hormone analogue [Leu13]motilin has been produced in high yield by recombinant DNA techniques. The peptide was expressed from a multicopied [Leu13]motilin gene fused to a salmon growth hormone gene fragment. The monomeric [Leu13]motilin was obtained by treatment of the fusion protein with cyanogen bromide, carboxypeptidase A and B. [Leu13]motilin showed the equivalent biological activity to that of the natural form.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01027570
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  • 8
    Electronic Resource
    Electronic Resource
    The duality of teleost gonadotropins (1989)
    Springer
    Fish physiology and biochemistry 7 (1989), S. 29-38 
    Details
    ISSN: 1573-5168
    Keywords: gonadotropins ; isolation ; amino acid sequence ; immunocytochemistry ; steroid activities ; GnRH
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The duality of salmon gonadotropins has been proved by biochemical, biological, and immunological characterization of two chemically distinc gonadotropins. GTH I and GTH II were equipotent in stimulating estradiol production, whereas GTH II appears to be more potent in stimulating maturational steroid synthesis. The ratio of plasma levels and pituitary contents of GTHs and the secretory control by a GnRH suggest that GTH I is the predominant GTH during vitellogenesis and early stages of spermatogenesis in salmonids, whereas GTH II is predominant at the time of spermiation and ovulation. GTH I and GTH II are found in distinctly separate cells. In trout, GTH I is expressed first in ontogeny, whereas GTH II cells appear coincident with the onset of spermatogenesis and vitellogenesis, and increase dramatically at the time of final reproductive maturation. Comparison of the amino acid sequences of polypeptides and the base sequences of cDNA revealed that salmon GTH I β is more similar to bovine FSHβ than bovine LHβ and salmon GTH II β shows higher homology to bovine LHβ than to bovine FSHβ. The existence of two pituitary gonadotropins in teleosts as well as tetrapods suggests that the divergence of the GTH gene took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00004687
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  • 9
    Electronic Resource
    Electronic Resource
    Expression of salmon growth hormone in the cyanobacteriumAgmenellum quadruplicatum (1991)
    Springer
    Biotechnology letters 13 (1991), S. 851-856 
    Details
    ISSN: 1573-6776
    Source: Springer Online Journal Archives 1860-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary The salmon growth hormone gene was introduced into the cyanobactriumAgmenellum quadruplicatum PR-6 by plasmid transformation. The gene expressed the hormone under thetrp promoter ofEscherichia. coli. The amount was estimated to be approximately 0.1% of the total cell protein.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01022085
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  • 10
    Electronic Resource
    Electronic Resource
    Effect of N-terminal deletion of signal peptide on the secretion of human granulocyte-colony stimulating factor in mammalian cells (1990)
    Springer
    Biotechnology letters 12 (1990), S. 87-91 
    Details
    ISSN: 1573-6776
    Source: Springer Online Journal Archives 1860-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary The nine N-terminal amino acids of signal peptide differ between human and mouse granulocyte-colony stimulating factors (G-CSF). To study the function of this non-conserved sequence, cDNA lacking the sequence has been constructedin vitro. The deleted signal peptide promoted the secretion of hG-CSF in CHO cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01022421
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