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 degrees 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.
Patel
C N
CN
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Lind
M C
MC
Pielak
G J
GJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000096:biosynthesis / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005813:Genes, Synthetic; D006736:Horses; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics
D003574:Cytochrome c Group; D011994:Recombinant Proteins