Expression of outer mitochondrial membrane cytochrome b5 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 10(4) 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).
Sergeev
G V
GV
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk, 220141, Belarus.
Gilep
A A
AA
Estabrook
R W
RW
Usanov
S A
SA
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D015786:Cytochromes b5 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D051379:Mice; D051336:Mitochondrial Membranes / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D014357:Trypsin / Q000378:metabolism
D008024:Ligands; D024101:Mitochondrial Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D015786:Cytochromes b5; D009251:NADPH-Ferrihemoprotein Reductase; D014357:Trypsin