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Heterologous expression of NADPH:cytochrome P450 reductase
Stráňava, Martin ; Černá, Věra (advisor) ; Martínek, Václav (referee)
NADPH:cytochrome P450 reductase (CPR) is a 78 kDa flavoprotein, which is together with cytochrome P450 component of monooxygenase system bound in the membrane of the endoplasmic reticulum. Monooxygenase system is involved in the metabolism of a wide range of organic substances, including drugs or various pollutants present in the environment (polycyclic aromatic hydrocarbons, aromatic amines, etc.). CPR works as a transporter of reducing equivalents from NADPH to the cytochromes P450. For proper interaction with cytochromes P450, intact N-terminal hydrophobic domain anchoring protein in the membrane is needed. Removing this domain, e.g. during trypsin proteolysis, gives rise a soluble CPR (72 kDa) and cause loss of catalytic activity towards cytochrome P450. During heterologous expression in E. coli proteolytically sensitive site of CPR (Lys56 - Ile57) is cleaved by intracellular trypsin-like proteases, that may negatively affect the yields of native 78 kDa protein. This thesis describes the heterologous expression, purification and characterization of two forms of rat CPR. WtCPR is a protein naturally occurring in rats (Wistar strain), while mCPR contains one amino acid substitution (K56Q) in the site of proteolytic degradation. The result of that substitution is proteolytically stable CPR,...
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Heterologous expression of NADPH:cytochrome P450 reductase
Stráňava, Martin ; Černá, Věra (advisor) ; Martínek, Václav (referee)
NADPH:cytochrome P450 reductase (CPR) is a 78 kDa flavoprotein, which is together with cytochrome P450 component of monooxygenase system bound in the membrane of the endoplasmic reticulum. Monooxygenase system is involved in the metabolism of a wide range of organic substances, including drugs or various pollutants present in the environment (polycyclic aromatic hydrocarbons, aromatic amines, etc.). CPR works as a transporter of reducing equivalents from NADPH to the cytochromes P450. For proper interaction with cytochromes P450, intact N-terminal hydrophobic domain anchoring protein in the membrane is needed. Removing this domain, e.g. during trypsin proteolysis, gives rise a soluble CPR (72 kDa) and cause loss of catalytic activity towards cytochrome P450. During heterologous expression in E. coli proteolytically sensitive site of CPR (Lys56 - Ile57) is cleaved by intracellular trypsin-like proteases, that may negatively affect the yields of native 78 kDa protein. This thesis describes the heterologous expression, purification and characterization of two forms of rat CPR. WtCPR is a protein naturally occurring in rats (Wistar strain), while mCPR contains one amino acid substitution (K56Q) in the site of proteolytic degradation. The result of that substitution is proteolytically stable CPR,...
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