PMID: 18707589Aug 19, 2008Paper

Engineering, expression, and purification of "soluble" human cytochrome P45017alpha and its functional characterization

Biochemistry. Biokhimii︠a︡
Tatiana A PechurskayaS A Usanov

Abstract

To engineer a "soluble" form of membrane-bound cytochrome P45017alpha (CYP17)--a key enzyme in steroid hormone biosynthesis--in the present work we have built a computer model of the tertiary structure of the hemeprotein, identified the surface hydrophobic amino acid residues, substituted these residues for more hydrophilic ones, and expressed and purified hydrophilized forms of CYP17. We have constructed and purified the following mutant forms of human CYP17: CYP17dH (CYP17 with deleted hydrophobic N-terminal sequence (Delta(23))) and CYP17mod (CYP17dH with substituted cluster of hydrophobic amino acid residues in the region of the FG-loop). Removal of the N-terminal sequence responsible for interaction with the membrane does not dramatically change the association of the protein with the membrane. However, CYP17mod containing hydrophilic FG-loop is mostly localized in the cytosolic fraction. Thus, in the present work we for the first time engineered a "soluble" form of the usually membrane-bound human CYP17 that is not bound to membrane. The expression degree of CYP17mod is approximately 900 nmol/liter of culture. The hemeprotein can be purified to apparent homogeneity without using detergents at any purification step. It is ...Continue Reading

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Citations

Apr 27, 2013·The Journal of Biological Chemistry·D Fernando EstradaEmily E Scott
Dec 5, 2013·Biochemical and Biophysical Research Communications·Yogan KhatriStephen G Sligar
Jun 27, 2020·Biomedit︠s︡inskai︠a︡ khimii︠a︡·P I KorolevaV V Shumyantseva
Jul 28, 2020·Drug Metabolism and Personalized Therapy·Anna A MakhovaVictoria V Shumyantseva
Jun 7, 2021·The FEBS Journal·Philip HartzRita Bernhardt
Dec 4, 2021·Drug Metabolism and Personalized Therapy·Victoria V ShumyantsevaSergei A Usanov

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