Expression of Functionally Active Cytochrome b₅ in Escherichia coli: Isolation, Purification, and Use of the Immobilized Recombinant Heme Protein for Affinity Chromatography of Electron-Transfer Proteins

M. V. Chudaev¹ and S. A. Usanov^1,2

¹Institute of Bioorganic Chemistry, Academy of Sciences of Belarus, ul. Zhodinskaya 5, Minsk, 220141 Belarus; fax: (375-172) 63-72-74; E-mail: usanov@eco2.iasnet.com

²To whom correspondence should be addressed.

Submitted November 22, 1996.

Cytochrome b₅ is an integral membrane protein which is localized in endoplasmic reticulum membranes. In this paper we present the results on expression in E. coli, purification, and characterization of recombinant rat cytochrome b₅. The full-length cDNA for rat liver microsomal cytochrome b₅ has been modified using polymerase chain reaction (PCR) to introduce corresponding restriction sites as well as to insert silent mutations in the N-terminal sequence to increase the content of A and T nucleotides that prevents formation of elements of secondary structure of the mRNA transcripts and facilitates high expression. The expression plasmid was constructed by cloning of amplified cDNA to pCWori+ plasmid and used for transformation of E. coli DH5alpha. The optimization of recombinant cytochrome b₅ expression procedure induces expression level up to 3000 nmoles per liter of growth medium; this confers in the cells a deep pink color. The most interesting fact is that cytochrome b₅ is expressed in this system in the reduced state. Recombinant cytochrome b₅ was purified from solubilized cell membranes by a combination of ion-exchange chromatography and gel filtration. During purification, part of the cytochrome b₅ is subjected to limited proteolysis with formation of a truncated form. Sequencing of the N-terminal part of the recombinant cytochrome b₅ indicates that it coincides with the sequence of rat cytochrome b₅. Recombinant cytochrome b₅ was found to have physicochemical, catalytic, and immunochemical properties very similar to that of the native protein and was used as an efficient affinity matrix for purification of the various electron-transfer proteins.

KEY WORDS: cytochrome b₅, expression in Escherichia coli, ion-exchange chromatographic purification, affinity chromatography.

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