The large subunit of herpes simplex virus type 1 ribonucleotide reductase: expression in Escherichia coli and purification.
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.
Furlong
J
J
MRC Virology Unit, Glasgow, Scotland.
Conner
J
J
McLauchlan
J
J
Lankinen
H
H
Galt
C
C
Marsden
H S
HS
Clements
J B
JB
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D012264:Ribonucleotide Reductases