Characterization of Horse Cytochrome c Expressed in Escherichia coli

We have expressed horse cytochrome c in Escherichia coli. The gene was designed with E. coli codon bias and assembled by using a recursive polymerase chain reaction method. The far-ultraviolet and near-ultraviolet/Soret circular dichroism (CD) spectra show that the structure of recombinant horse cytochrome c is the same as that of the authentic protein. CD-detected thermal denaturation studies were used to measure the thermodynamic parameters associated with two-state denaturation. The free energy of denaturation for the recombinant protein is 10.0 ± 2.3 kcal mol−1 at pH 4.6 and 25°C, which agrees with the value for the authentic protein. The expression system will help advance our understanding of the roles of cytochrome c in electron transfer, oxidative stress, and apoptosis by allowing the production of protein variants.