Cloning and expression of the gene encoding the soluble cytochrome b₅₆₂ of Escherichia coli

The gene for the soluble cytochrome b₅₆₂ from Escherichia coli B has been cloned on a SalI fragment. The analysis of the gene reveals the presence of a leader sequence in front of the sequence encoding the mature protein. Expression of cytochrome b₅₆₂ using the lac‐promoter produced the protein to a level of 3–5% of total protein. This over‐production enables employment of a simple, high‐yield purification protocol to obtain homogeneous cytochrome b₅₆₂. Spectroscopic and N‐terminal sequence analyses of the purified protein demonstrate that it is identical to the chromosomally expressed cytochrome b₅₆₂ purified and characterized from E. coli B [Itagaki, E. & Hager, L. P. (1966) J. Biol. Chem. 241, 3687–3695]. It is demonstrated that the genomic sequence codes for a classic N‐terminal signal sequence and that mature cytochrome b₅₆₂ is translocated to the periplasmic space.