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,...
Identifer | oai:union.ndltd.org:nusl.cz/oai:invenio.nusl.cz:307847 |
Date | January 2012 |
Creators | Stráňava, Martin |
Contributors | Černá, Věra, Martínek, Václav |
Source Sets | Czech ETDs |
Language | Czech |
Detected Language | English |
Type | info:eu-repo/semantics/masterThesis |
Rights | info:eu-repo/semantics/restrictedAccess |
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