The large subunit of herpes simplex virus type 1 ribonucleotide reductase: expression in Escherichia coli and purification

Furlong, J., Conner, J., McLauchlan, J. , Lankinen, H., Galt, C., Marsden, H.S. and Clements, J.B. (1991) The large subunit of herpes simplex virus type 1 ribonucleotide reductase: expression in Escherichia coli and purification. Virology, 182(2), pp. 846-851. (doi: 10.1016/0042-6822(91)90627-N)

Abstract

The open reading frame of the large subunit (R1) of herpes simplex virus type 1 (HSV-1) ribonucleotide reductase has been positioned downstream of the phage T7 gene 10 promoter in the expression vector, pET. Transformation of this recombinant plasmid into Escherichia coli BL21 DE3 cells containing the T7 RNA polymerase, under the control of the lac UV5 promoter, allows expression of the subunit on induction of the T7 RNA polymerase by isopropyl thiodigalactoside. The expressed protein is soluble and can be purified with yields up to 0.5 mg of R1 per litre of bacterial culture. The subunit can complement R2 produced in BHK cells or E. coli to give specific activities comparable to that produced in BHK cells infected with HSV-1. Enzyme activity reconstituted from E. coli-expressed R1 and R2 is inhibited by the nonapeptide YAGAVVNDL with an IC50 comparable to that obtained with enzyme extracted from BHK cells infected with HSV-1. Results suggest that the E. coli produced enzyme is a good source of protein for further structural and functional studies.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: McLauchlan, Professor John
• Authors: Furlong, J., Conner, J., McLauchlan, J., Lankinen, H., Galt, C., Marsden, H.S., and Clements, J.B.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity; College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research
• Journal Name: Virology
• Publisher: Elsevier
• ISSN: 0042-6822
• ISSN (Online): 1096-0341