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1

Expression of soluble recombinant TGF-beta type II receptor fused with the Fc portion of human IgG1 [sTbetaRII-Fc] in NS0 cells

100%
Kwiatkowska E. P., Kazimierczak U., Mackiewicz A., Kowalczyk D. W.
Acta Biochimica Polonica
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2006
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tom 53
|
nr 2
We have constructed and expressed recombinant chimeric soluble TGF-β type II receptor fused with the Fc portion of human IgG1 (sTβRII-Fc) in NS0 mouse myeloma cells and isolated cell lines constitutively secreting very high levels of biologically active protein. The GS-NS0 expression system takes advantage of the strong human cytomegalovirus immediate early promoter expression vector and glutamine synthetase as a selectable marker. The recombinant chimeric receptor could be produced in high amounts and efficiently purified by one step chromatography on a protein A column. Biochemical studies revealed that recombinant sTβRII-Fc binds native TGF-β1 and TGF-β3 isoforms and neutralizes their activity in vitro.
2

Characterisation of a new molecule based on two E2 sequences from bovine viral diarrhoea-mucosal disease virus fused to the human immunoglobulin Fc fragment

86%
Gonzalez Pose A., Montesino Segui R., Maura Perez R., Hugues Salazar F., Cabezas Avila I., Altamirano Gomez C., Sanchez Ramos O., Toledo J. R.
Journal of Veterinary Research
|
2021
|
tom 65
|
nr 1
3

Thermostable Pyrococcus woesei beta-D-galactosidase - high level expression, purification and biochemical properties

86%
Wanarska M., Kur J., Pladzyk R., Turkiewicz M.
Acta Biochimica Polonica
|
2005
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tom 52
|
nr 4
The gene encoding β-D-galactosidase from Pyrococcus woesei was PCR amplified, cloned, expressed in Escherichia coli under the control of an inducible T7 promoter, purified and characterized. The expression system was developed by the construction of recombinant plasmid, based on the high copy number pUET1 vector, giving four times more efficient expression of P. woesei β-D-galactosidase (20 mg of enzyme from 1 liter of culture) than that obtained from a previously constructed one. The recombinant enzymes were purified in a two-step procedure: double heat-denaturation of E. coli cell proteins and affinity chromatography on p-aminobenzyl 1-thio-β-D-galactopyranoside-agarose. To achieve efficient purification of P. woesei β-D-galactosidase by immobilized metal-ion affinity chromatography (IMAC), a His-tag was placed either at the N- or the C-terminal of the coding sequence. The obtained fusion proteins revealed the same specific activity of approximately 5400 U/mg, which was 10 times lower than the wild-type β-D-galactosidase (51100 U/mg). The activity of P. woesei β-D-galactosidase was enhanced by thiol compounds, Mg2+ ions and D-galactose, and was inhibited by heavy metal ions and D-glucose, while Ca2+ ions had no effect.
4

Expression of genes 51, 27, 28 coding for proteins of the central part of bacteriophage T4 baseplate in the bacteriophage T7 promoter-RNA polymerase expression system

86%
Nieradko J., Podgorska B.
Acta Biochimica Polonica
|
1993
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tom 40
|
nr 2
A fragment of T4 DNA (XbaI-HindIII) comprising the genes 51,27,28, which encodes the central plug proteins was cloned into plasmid pT7-5 and p7-6 (T7 RNA polymerase expressing system). The examined genes were only overexpressed when the orientation of cloned DNA to promoter <1>10 was as follows: promoter 10 and genes 51, 27, 28. This was achieved when the fragment (Xbal-Hindlll) was cloned into plasmid pT7-5. Gene 27 and 28 were overexpressed when the intact fragment (Xbal-Hindlll) was used. The high rate of the synthesis of proteins 27 and/or 28 had a strong inhibitory effect on the level of synthesis of the product of gene 51. For the overexpression of gene 51 in this system a deletion derivate which was devoid of gene 28 and a larger fragment of gene 27 was prepared.
5

Recent developments in recombinant proteins for diagnosis of human fascioliasis

72%
Mirzadeh A., Jafarihaghighi F., Kazemirad E., Sabzevar S. S., Tanipour M. H., Ardjmand M.
Acta Parasitologica
|
2021
|
tom 66
|
nr 1
6

Alternative insect cell lines for foreign protein production using baculovirus expression system

72%
Olejnik A., Grajek W.
Folia Histochemica et Cytobiologica. Supplement
|
1997
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tom 35
|
nr 2
7

Expression of Bombyx mori nucleopolyhedrovirus ORF76 in permissive and non-permissive cell lines by a novel Bac-to-Bac-BmNPV baculovirus expression system

58%
Su W. J., Wu Y., Wu H.-L., Zhu S.-Y., Wang W.-B.
Polish Journal of Microbiology
|
2008
|
tom 57
|
nr 4
Open reading frame 76 of Bombyx mori nucleopolyhedrovirus (BmNPV), designated as Bm76, is a gene whose function is completely unknown. With EGFP fused to the 3' terminal of Bm76 as the reporter gene and BmNPV bacmid as the expression vector, a recombinant bacmid was successfully constructed expressing Bm76-EGFP fusion protein under the control of polyhedrin promoter in Bombyx mori cells (Bm cells), BmNPV's permissive cell line, laying the foundation for rescue experiment of Bm76 deletion mutant. Moreover, the supernatant from Bm cells transfected with the recombinant bacmid was used to infect Trichoplusia Ni cells (Tn cells), BmNPV's non-permissive cell line. Unexpectedly, the expression of Bm76-EGFP fusion protein in some Tn cells was detected, implying that viral DNA was replicated in these cells. The causes are being studied for the inability of BmNPV to produce enough viable budded viruses in Tn cells despite of viral DNA replication.
8

Molecular cloning and expresion in Escherichia coli of a gene coding for bovine S100A1 protein and its Glu32-Gln and Glu73-Gln mutants

58%
Bolewska K., Kozlowska H., Groch G., Mikolajek B., Bierzynski A.
Acta Biochimica Polonica
|
1997
|
tom 44
|
nr 2
Calcium binding S100A1 protein consists of two S100 alpha subunits. On the basis of sequence homology to other S100 proteins it is believed that the binding loops are formed by amino-acid residues 19-32 and 62-73 of S100 alpha polypeptide chain. In the oxidized form of the protein the subunits are linked covalently with each other by a disulphide bond between their Cys85 residues. A synthetic gene coding for bovine S100 alpha subunit was constructed and cloned into a derivative of pAED4 plasmid. The gene was expressed in Escherichia coli utilizing the T7 expression system. The expression products were purified and identified using mass spectrometry and by sequencing of their N- and C-termini. Three different forms (a, b, and c) of S100 alpha were produced: with the native sequence, with the initiator methionine at the N-terminus, and with an additional alanine at the C-terminus as well as with the initiator methionine. The material was partly oxidized. Interestingly, only the homodimers of a, b, and c species were formed. The total yield of the protein was about 50 mg/l of culture. Genes coding for Glu32-->Gln and Glu73-->Gln mutants of S100 alpha were obtained by site-directed mutagenesis and expressed in the same system. In both cases similar mixtures of oxidized and reduced a, b, and c species have been obtained. The total yield of E73Q mutant is similar to that of the native protein and that of E32Q lower by about a half. As expected, the mutants of S100 alpha subunits bind only one calcium ion.
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