Expression of Outer Mitochondrial Membrane Cytochrome b₅ in Escherichia coli. Purification of the Recombinant Protein and Studies of Its Interaction with Electron-Transfer Partners

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G. V. Sergeev¹, A. A. Gilep¹, R. W. Estabrook², and S. A. Usanov^1*

¹Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, ul. Kuprevicha 5/2, 220141 Minsk, Belarus; fax: 375 (172) 63-7274; E-mail: usanov@iboch.bas-net.by

²Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX, 75235-9038, USA; E-mail: RonaldEstabrook@UTSouthwestern.edu

^* To whom correspondence should be addressed.

Received January 24, 2006; Revision received February 23, 2006

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In the present work, we report expression in Escherichia coli, purification, and characterization of recombinant full-length cytochrome b₅ from outer mitochondrial membrane. Optimization of expression conditions for cytochrome b₅ from outer mitochondrial membrane allowed reaching expression level up to 10⁴ nmol of the hemeprotein per liter of culture. Recombinant cytochrome b₅ from outer mitochondrial membrane was purified from cell lysate by using metal-affinity chromatography. It has physicochemical, spectral, and immunochemical properties similar to those of cytochrome b₅ from rat liver outer mitochondrial membrane. Immobilized recombinant mitochondrial cytochrome b₅ was used as affinity ligand to study its interaction with electron transfer proteins. By using this approach, it is shown that in interaction of NADPH:cytochrome P450 reductase with both forms of cytochrome b₅ an important role is played by hydrophobic interactions between proteins, although the contribution of these interactions in complex formation with NADPH:cytochrome P450 reductase is different for isoforms of cytochrome b₅.

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KEY WORDS: microsomal cytochrome b₅, cytochrome b₅ from outer mitochondrial membrane, expression in Escherichia coli, affinity chromatography, purification

DOI: 10.1134/S0006297906070121

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