EXPRESSION OF FUNCTIONALLY ACTIVE CYTOCHROME B(5) IN ESCHERICHIA-COLI- ISOLATION, PURIFICATION, AND USE OF THE IMMOBILIZED RECOMBINANT HEME PROTEIN FOR AFFINITY-CHROMATOGRAPHY OF ELECTRON-TRANSFER PROTEINS

Cytochrome b(5) 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 recomb inant rat cytochrome b(5). The full-length cDNA for rat liver microsom al cytochrome b(5) 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 o f A and T nucleotides that prevents formation of elements of secondary structure of the mRNA transcripts and facilitates high expression. Th e expression plasmid was constructed by cloning of amplified cDNA to p CWori+ plasmid and used for transformation of E. coli DH5 alpha. The o ptimization of recombinant cytochrome b(5) expression procedure induce s 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 t hat cytochrome b(5) is expressed in this system in the reduced slate. Recombinant cytochrome b(5) was purified from solubilized cell membran es by a combination of ion-exchange chromatography and gel filtration. During purification, part of the cytochrome b(5) is subjected to limi ted proteolysis with formation of a truncated form. Sequencing of the N-terminal part of the recombinant cytochrome b(5) indicates that it c oincides with the sequence of rat cytochrome b(5). Recombinant cytochr ome b(5) was found to have physicochemical, catalytic, and immunochemi cal properties very similar to that of the native protein and was used as an efficient affinity matrix for purification of the various elect ron-transfer proteins.