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Yeast cytochrome c peroxidase expression in Escherichia coli and rapid isolation of various highly pure holoenzymes.
- Source :
-
Protein expression and purification [Protein Expr Purif] 2000 Jun; Vol. 19 (1), pp. 139-47. - Publication Year :
- 2000
-
Abstract
- 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.<br /> (Copyright 2000 Academic Press.)
- Subjects :
- Amino Acid Sequence
Apoenzymes chemistry
Apoenzymes genetics
Apoenzymes isolation & purification
Chromatography, High Pressure Liquid
Cytochrome-c Peroxidase chemistry
Cytochrome-c Peroxidase genetics
Electrophoresis, Polyacrylamide Gel
Escherichia coli enzymology
Escherichia coli genetics
Holoenzymes chemistry
Holoenzymes genetics
Holoenzymes isolation & purification
Magnetic Resonance Spectroscopy
Mass Spectrometry
Molecular Sequence Data
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins isolation & purification
Sequence Analysis, Protein
Spectrophotometry, Ultraviolet
Cytochrome-c Peroxidase isolation & purification
Saccharomyces cerevisiae chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1046-5928
- Volume :
- 19
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Protein expression and purification
- Publication Type :
- Academic Journal
- Accession number :
- 10833401
- Full Text :
- https://doi.org/10.1006/prep.2000.1220