Yeast cytochrome c peroxidase expression in Escherichia coli and rapid isolation of various highly pure holoenzymes.
A more efficient 2-day isolation and purification method for recombinant yeast cytochrome c peroxidase produced in Escherichia coli is presented. Two types of recombinant "wild-type" CcP have been produced and characterized, the recombinant nuclear gene sequence and the 294-amino-acid original protein sequence. These two sequences constitute the majority of the recombinant "native" or wild-type CcP currently in production and from which all recombinant variants now derive. The enzymes have been subjected to extensive physical characterizations, including sequencing, UV-visible spectroscopy, HPLC, gel electrophoresis, kinetic measurements, NMR spectroscopy, and mass spectrometry. Less extensive characterization data are also presented for recombinant, perdeuterated CcP, an enzyme produced in >95% deuterated medium. All of these results indicate that the purified recombinant wild-type enzymes are functionally and spectroscopically identical to the native, yeast-isolated wild-type enzyme. This improved method uses standard chromatography to produce highly purified holoenzyme in a more efficient manner than previously achieved. Two methods for assembling the holoenzyme are described. In one, exogenous heme is added at lysis, while in the other heme biosynthesis is stimulated in E. coli. A primary reason for developing this method has been the need to minimize loss of precious, isotope-labeled enzyme and, so, this method has also been used to produce both the perdeuterated and the (15)N-labeled enzyme, as well as several variants.
Teske
J G
JG
Department of Chemistry, Washington State University, Pullman, Washington 99164-4630, USA.
Savenkova
M I
MI
Mauro
J M
JM
Erman
J E
JE
Satterlee
J D
JD
D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D003578:Cytochrome-c Peroxidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D020539:Sequence Analysis, Protein; D013056:Spectrophotometry, Ultraviolet
D001051:Apoenzymes; D020035:Holoenzymes; D011994:Recombinant Proteins; D003578:Cytochrome-c Peroxidase