title: Improved system for heterologous expression of cytochrome c mutants in Escherichia coli.
abstract: We improved an already existing cytochrome c expression system to a reliable, tightly controllable one to achieve a higher expression yield for single cysteine mutants of horse cytochrome c. The protein is heterologously overexpressed in E. coli together with the maturation coordinating enzyme heme lyase from yeast. Various plasmid constructs and host strains were tested for protein expression yield and routinely around 35 mg/L yield was achieved, which is a good result for a post-translationally modified enzyme. The purpose of producing cysteine mutants is to position accessible cysteine residues on the surface of cytochrome c which can be labeled with a photoactive redox dye, 8-thiouredopyrene-1,3,6-trisulfonate, TUPS. TUPS labeled proteins have been used for intramolecular and intermolecular electron transfer measurements. Here, we initiate the photoreduction of cytochrome c oxidase, the natural electron acceptor partner of cytochrome c by an appropriate cytochrome c mutant labeled with TUPS. The electron transfer from cytochrome c to the first cytochrome oxidase redox cofactor, copper A, is shown to be very fast.
authors.affiliation: Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: Katalin
authors.initials: K
authors.lastname: Tenger
authors.forename: P
authors.initials: P
authors.lastname: Khoroshyy
authors.forename: K L
authors.initials: KL
authors.lastname: Kovács
authors.forename: L
authors.initials: L
authors.lastname: Zimányi
authors.forename: G
authors.initials: G
authors.lastname: Rákhely
mesh_terms: D001483:Base Sequence; D045304:Cytochromes c / Q000235:genetics*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013057:Spectrum Analysis
chemical_list: D017931:DNA Primers; D045304:Cytochromes c