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Expression of outer mitochondrial membrane cytochrome b 5 in Escherichia coli. Purification of the recombinant protein and studies of its interaction with electron-transfer partners

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Abstract

In the present work, we report expression in Escherichia coli, purification, and characterization of recombinant full-length cytochrome b 5 from outer mitochondrial membrane. Optimization of expression conditions for cytochrome b 5 from outer mitochondrial membrane allowed reaching expression level up to 104 nmol of the hemeprotein per liter of culture. Recombinant cytochrome b 5 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 b 5 from rat liver outer mitochondrial membrane. Immobilized recombinant mitochondrial cytochrome b 5 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 b 5 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 b 5.

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Abbreviations

DTT:

dithiothreitol

IPTG:

isopropyl-β-thiogalactopyranoside

PMSF:

phenylmethylsulfonyl fluoride

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Authors and Affiliations

  1. Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, ul. Kuprevicha 5/2, 220141, Minsk, Belarus

    G. V. Sergeev, A. A. Gilep & S. A. Usanov

  2. Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX, 75235-9038, USA

    R. W. Estabrook

Authors
  1. G. V. Sergeev
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  2. A. A. Gilep
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  3. R. W. Estabrook
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  4. S. A. Usanov
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Correspondence to S. A. Usanov.

Additional information

Published in Russian in Biokhimiya, 2006, Vol. 71, No. 7, pp. 972–983.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM06-014, April 16, 2006.

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Sergeev, G.V., Gilep, A.A., Estabrook, R.W. et al. Expression of outer mitochondrial membrane cytochrome b 5 in Escherichia coli. Purification of the recombinant protein and studies of its interaction with electron-transfer partners. Biochemistry (Moscow) 71, 790–799 (2006). https://doi.org/10.1134/S0006297906070121

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  • DOI: https://doi.org/10.1134/S0006297906070121

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