Expression and Purification of the Amphipathic Form of Rabbit Cytochrome b₅ in Escherichia coli

Abstract

Cytochrome b₅ (cyt b₅) is an electron transfer protein that exists in a membrane-bound form in the endoplasmic reticulum where it is anchored to the membrane via a carboxyl-terminal transmembrane α-helix (1–3). The membrane-bound form of cyt b₅ provides reducing equivalents for the biosynthesis of a variety of lipids including unsaturated fatty acids, plasmalogens, and cholesterol. In addition, it facilitates the cytochrome P450 catalyzed oxidation of selected substrates (2). The membrane domain is linked to the aminoterminal catalytic heme-containing domain via an 11 amino acid linker. The mammalian cyts b₅ are typically greater than 90% similar in sequence and may be interchangeable in some systems (4). Nevertheless, our laboratory uses rabbit cytochrome P450 2B4 and cytochrome P450 reductase and has elected to use the rabbit cyt b₅ so all proteins are from a single species. Cyt b₅ also exists in a soluble form in red blood cells where it functions to maintain hemoglobin in its ferrous oxygen-carrying form (5).