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Expression of three diadinoxanthin de-epoxidase genes of Phaeodacylum tricornutum in Escherichia coli Origami b and BL21 strain

100%

Bojko M., Olchawa-Pajor M., Tuleja U., Kuczyńska P., Strzałka W., Latowski D., Strzałka K.

2013

vol. 60

issue 4

857-860

In the diadinoxanthin cycle the epoxy group is removed from diadinoxanthin and diatoxanthin is created. This conversion takes place e.g. in diatoms with the involvement of the enzyme diadinoxanthin de-epoxidase. In one of the diatom species, Phaeodactylum tricornutum (CCAP 1055/1 strain with genome sequenced) three de-epoxidase genes (PtVDE, PtVDL1, PtVDL2) have been identified, but only one of them (PtVDE) corresponds to violaxanthin de-epoxidase, an enzyme which is commonly found in higher plants. In these studies, the expression of two de-epoxidase genes of another Phaeodactylum tricornutum strain (UTEX 646), which is commonly used in diatom studies, were obtained in Origami b and BL21 E. coli strains. The molecular masses of the mature proteins are about 49 kDa and 60 kDa, respectively, for VDE and VDL2. Both enzymes are active with violaxanthin as a substrate.

Selection and analysis of a DNA aptamer binding α-amanitin from Amanita phalloides

84%

Muszyńska K., Ostrowska D., Bartnicki F., Kowalska E., Bodaszewska-Lubaś M., Hermanowicz P., Faulstich H., Strzałka W.

2017

vol. 64

issue 3

401-406

Mushroom foraging is very popular in some regions of the world. Sometimes toxic and edible mushrooms are mistaken by mushroom collectors, leading to serious human poisoning. The group of mushrooms highly dangerous for human health includes Amanita phalloides. This mushroom produces a toxic octapeptide called α-amanitin which is an inhibitor of nuclear RNA polymerase II. The inhibition of this polymerase results in the abortion of mRNA synthesis. The ingestion of A. phalloides causes liver failure due to the fact that most of the toxin is uptaken by hepatocytes. The hospitalization of poisoned patients involves the removal of the toxin from the digestive tract, its dilution in the circulatory system and the administration of therapeutic adjuvants. Since there is no effective antidote against amanitin poisoning, in this study we developed a DNA aptamer exhibiting specific binding to α-amanitin. This aptamer was selected using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method. Next, its ability of toxin removal from aqueous solution was confirmed by pull-down assay. The aptamer region sufficient for α-amanitin binding was determined. Finally, the dissociation constant of the α-amanitin/DNA aptamer complex was calculated.

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2 Acta Biochimica Polonica

1 2017

1 2013

2 Strzałka W.

1 Bartnicki F.

1 Bodaszewska-Lubaś M.

1 Bojko M.

1 Faulstich H.

1 Hermanowicz P.

1 Kowalska E.

1 Kuczyńska P.

1 Latowski D.

1 Muszyńska K.

1 Olchawa-Pajor M.

1 Ostrowska D.

1 Strzałka K.

1 Tuleja U.

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Expression of three diadinoxanthin de-epoxidase genes of Phaeodacylum tricornutum in Escherichia coli Origami b and BL21 strain

Selection and analysis of a DNA aptamer binding α-amanitin from Amanita phalloides