Vaccinia virus ribonucleotide reductase expression and isolation of the recombinant large subunit.
The vaccinia virus gene encoding the 87-kDa protein that comprises the large subunit of ribonucleotide reductase (vvR1) was cloned into a bacterial expression vector under the control of an inducible promoter. Culture of Escherichia coli cells harboring the recombinant plasmid under standard induction conditions (0.4 mM isopropyl beta-D-thiogalactopyranoside, 37 degrees C) resulted in synthesis of a completely insoluble product. Production of soluble vvR1 was achieved by growing bacteria at low temperature (15 degrees C) during the induction period, initiating induction at low cell density, and using a low concentration (0.05 mM) of the inducer isopropyl beta-D-thiogalactopyranoside. Hydroxyurea, an inhibitor of ribonucleotide reductase, increased production of soluble vvR1 in a dose-dependent manner. Recombinant vvR1 was purified from a high salt extract of the E. coli lysate in four steps, the last utilizing an affinity column consisting of the carboxyl-terminal seven amino acids of the vvR2 protein linked to an insoluble resin. Using purified recombinant vvR2 to reconstitute active enzyme, we determined that maximizing the rate of CDP reduction required pH 8.0-8.8, 50 mM dithiothreitol, and 2 mM ATP. Specific activity of purified vvR1 was 122 nmol/min/mg. Limited proteolysis of the vvR1 protein revealed protease-resistant fragments approximately 30 and 58 kDa in size. To our knowledge, this study represents the first expression, solubilization, and isolation of a recombinant "eukaryotic" form of ribonucleotide reductase large subunit.
Slabaugh
M B
MB
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331-7305.
Davis
R E
RE
Roseman
N A
NA
Mathews
C K
CK
D001483:Base Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004790:Enzyme Induction; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006918:Hydroxyurea / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012264:Ribonucleotide Reductases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012264:Ribonucleotide Reductases; D006918:Hydroxyurea