Purification and characterization of the herpes simplex virus type 1 ribonucleotide reductase small subunit following expression in Escherichia coli.
The herpes simplex virus type 1 (HSV-1) gene encoding the ribonucleotide reductase (RR) small subunit (R2) was cloned as an unfused and intact open reading frame into a T7 RNA polymerase expression system in Escherichia coli. The expressed product was recovered from bacteria in soluble form and constituted 7% of the soluble protein. Protein purification yielded 3.5 mg of 95% pure R2 per litre of bacterial culture. The correct composition of the purified protein was verified by amino acid analysis and N-terminal sequencing. The isoelectric point of the protein was 5.3. Atomic emission spectroscopy indicated that the iron content of the E. coli-expressed R2 was 0.2 to 0.5 atoms of iron per R2 protomer as compared with a theoretical maximum value of 2. The E. coli-expressed HSV-1 R2 existed as a combination of a stable dimer and monomer. Combination of the E. coli-expressed R2 with the E. coli-expressed large subunit (R1) gave an active holoenzyme. Thus, the T7 expression system provides a rich source of enzymically active HSV-1 RR.
Lankinen
H
H
MRC Virology Unit, University of Glasgow, U.K.
McLauchlan
J
J
Weir
M
M
Furlong
J
J
Conner
J
J
McGarrity
A
A
Mistry
A
A
Clements
J B
JB
Marsden
H S
HS
D000596:Amino Acids / Q000032:analysis; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange / Q000379:methods; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D005800:Genes, Fungal; D005822:Genetic Vectors; D007525:Isoelectric Focusing / Q000379:methods; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D016366:Open Reading Frames; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics; D013604:T-Phages / Q000201:enzymology
D000596:Amino Acids; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012264:Ribonucleotide Reductases; D012321:DNA-Directed RNA Polymerases