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

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.