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Cloning and expression of human carbonyl reductase CR-1
Laštovková, Linda ; Wsól, Vladimír (advisor) ; Šimůnek, Tomáš (referee)
The purification of pOTB7 plasmid containing coding sequence of carbonyl reductase1 (CBR1) in cells of Escherichia coli (E. coli) was done by alkali hydrolysis. The sequence of CBR1 was multiplied by polymerase chain reaction (PCR) and synthesized primers with restriction sites were used. The left primer contained sequence for restrictive endonuclease NdeI and the right primer for restrictive endonuclease XhoI. Validation of the first step was confirmed by size measurement of the synthesized fragment with following restrictive analysis realized by restrictive endonuclease BamHI. Prepared sequence CBR1 was cloned into Topo vector, which was transformed into the competent E. coli cells. Topo vector was purified by alkali hydrolysis after innidation of E. coli cells. The size of cyclic Topo vector without CBR1 and Topo vector with CBR1 coding sequence was verified on agar gel by restrictive endonuclease BamHI. This was the validation of the second step. Subcloning of coding sequence CBR1 from Topo vector to express vector pET15b was the last step.
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Inhibition Study of Human Membrane-bound Carbonyl Reductase
Laštovková, Linda ; Wsól, Vladimír (advisor) ; Kvasničková, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Linda Laštovková Supervisor: prof. Ing. Vladimír Wsól, Ph.D. Title of thesis: Inhibition study of human membrane-bound carbonyl reductase. Carbonyl reductases are an important group of enzymes that participate in metabolism of both endogenous substances also xenobiotics. Potential anticancer drug oracin is one of such xenobiotics that is metabolised by various carbonyl reductases to two enantiomers of dihydrooracin. The new human microsomal carbonyl reductase also takes part in biotransformation of oracin. This enzyme was recently purified on Department of biochemical sciences (Faculty of Pharmacy in Hradec Králové). The aim of this study was to find some inhibitors of the new enzyme and distinguish it in terms of inhibitors from another microsomal carbonyl reductase 11β-hydroxysteroid dehydrogenase 1. Flavonoids are substances produced by plants, they have a different both positive and also negative effect on human organism. One of such effect is inhibition effect on diverse enzymes. Carbonyl reductases also fall in this group. It has been described inhibitory effect of different flavonoids on carbonyl reductases. Inhibition study of the new human micorosomal carbonylreductase was...
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Inhibition Study of Human Membrane-bound Carbonyl Reductase
Laštovková, Linda ; Wsól, Vladimír (advisor) ; Kvasničková, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Linda Laštovková Supervisor: prof. Ing. Vladimír Wsól, Ph.D. Title of thesis: Inhibition study of human membrane-bound carbonyl reductase. Carbonyl reductases are an important group of enzymes that participate in metabolism of both endogenous substances also xenobiotics. Potential anticancer drug oracin is one of such xenobiotics that is metabolised by various carbonyl reductases to two enantiomers of dihydrooracin. The new human microsomal carbonyl reductase also takes part in biotransformation of oracin. This enzyme was recently purified on Department of biochemical sciences (Faculty of Pharmacy in Hradec Králové). The aim of this study was to find some inhibitors of the new enzyme and distinguish it in terms of inhibitors from another microsomal carbonyl reductase 11β-hydroxysteroid dehydrogenase 1. Flavonoids are substances produced by plants, they have a different both positive and also negative effect on human organism. One of such effect is inhibition effect on diverse enzymes. Carbonyl reductases also fall in this group. It has been described inhibitory effect of different flavonoids on carbonyl reductases. Inhibition study of the new human micorosomal carbonylreductase was...
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Cloning and expression of human carbonyl reductase CR-1
Laštovková, Linda ; Wsól, Vladimír (advisor) ; Šimůnek, Tomáš (referee)
The purification of pOTB7 plasmid containing coding sequence of carbonyl reductase1 (CBR1) in cells of Escherichia coli (E. coli) was done by alkali hydrolysis. The sequence of CBR1 was multiplied by polymerase chain reaction (PCR) and synthesized primers with restriction sites were used. The left primer contained sequence for restrictive endonuclease NdeI and the right primer for restrictive endonuclease XhoI. Validation of the first step was confirmed by size measurement of the synthesized fragment with following restrictive analysis realized by restrictive endonuclease BamHI. Prepared sequence CBR1 was cloned into Topo vector, which was transformed into the competent E. coli cells. Topo vector was purified by alkali hydrolysis after innidation of E. coli cells. The size of cyclic Topo vector without CBR1 and Topo vector with CBR1 coding sequence was verified on agar gel by restrictive endonuclease BamHI. This was the validation of the second step. Subcloning of coding sequence CBR1 from Topo vector to express vector pET15b was the last step.
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