f Purification and characterization of the herpes simplex virus type 1 ribonucleotide reductase small subunit following expression in Escherichia coli
- Authors: Hilkka Lankinen, John McLauchlan, Malcolm Weir, Judy Furlong, Joe Conner, Anne McGarrity, Anil Mistry, J. Barklie Clements, Howard S. Marsden
- First Published Online: 01 June 1991, Journal of General Virology 72: 1383-1392, doi: 10.1099/0022-1317-72-6-1383
- Subject: Animal
- Issue Published:
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.
© Society for General Microbiology 1991 | Published by the Microbiology Society
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