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

Abstract

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. Copyright © 1991, Wiley Blackwell. All rights reserved