A Truncation of 2B Subfamily Cytochromes P450 Yields Increased Expression Levels, Increased Solubility, and Decreased Aggregation While Retaining Function

doi:10.1006/abbi.2001.2574

Archives of Biochemistry and Biophysics

Volume 395, Issue 1, 1 November 2001, Pages 57-68

https://doi.org/10.1006/abbi.2001.2574 Get rights and content

Abstract

The hydrophobic membrane-spanning domain in four cytochromes P450 2B was removed (Δ3-21) and several positive charges were substituted at the N-terminus to increase expression and solubility. Histidine residues were appended to the C-terminus to simplify purification. The truncated proteins were highly expressed in Escherichia coli, could be released from the membrane using high salt conditions, and were purified from this fraction to specific contents up to 19 nmol P450/mg protein using a single Ni²⁺-agarose column. Gel filtration revealed that truncated P450 2B1 forms a monodisperse solution of hexamers in the absence of detergent and >95% monomers in 0.25% sodium cholate. All truncated proteins, including human 2B6, were active with 7-ethoxy-4-trifluoromethylcoumarin, and truncated 2B1 was shown to retain the native regio- and stereospecificity of testosterone hydroxylation. These data demonstrate that modification of the N-terminus yields high levels of properly folded P450s 2B with increased solubility, which are suitable for functional and structural analysis.

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^☆: This work was supported by a National Research Service Award GM20674 (E.E.S.), ES03619 (J.R.H.), and Center Grant ES06676.

²: To whom correspondence should be addressed. Fax: (409) 772-9642. E-mail: [email protected].

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Regular ArticleA Truncation of 2B Subfamily Cytochromes P450 Yields Increased Expression Levels, Increased Solubility, and Decreased Aggregation While Retaining Function☆

Abstract

Biochem. Biophys. Res. Commun.

Arch. Bioch. Biophys.

Mol. Cell

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

J. Biol. Chem.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Biochem. Biophys. Res. Commun.

Curr. Drug Metab.

Drug Metab. Rev.

Drug Metab. Dispos.

Regular Article
A Truncation of 2B Subfamily Cytochromes P450 Yields Increased Expression Levels, Increased Solubility, and Decreased Aggregation While Retaining Function☆