High level expression in 293 cells of the herpes simplex virus type 2 ribonucleotide reductase subunit 2 using an adenovirus vector.
The herpes simplex viruses (HSV-1 and HSV-2) encode a ribonucleotide reductase consisting of two non-identical subunits (RR1 and RR2) which associate to form the active holoenzyme. To facilitate the purification and subsequent biochemical characterization of this enzyme, we have cloned the small subunit 2 of the HSV-2 ribonucleotide reductase (RR2HSV-2) in a helper-independent adenovirus type 5 vector under the control of the adenovirus type 2 major late promoter. After infection of 293 cells with the recombinant virus, the amount of RR2HSV-2 protein produced was eightfold higher than in HSV-2-infected cells. The specific activities of the RR2HSV-2 recombinant subunit and the RR2HSV-2 protein in HSV-2-infected cells were determined by their mixing with saturating amounts of isolated RR1HSV-1 subunit. By comparison of the relative amount of each RR2HSV-2 subunit with its specific activity, we calculated that the recombinant protein intrinsic activity was similar to that of the protein produced in HSV-2-infected cells. These results demonstrated that the adenovirus expression vector is a good system to produce an active RR2HSV-2 subunit in fairly high amounts.
Lamarche
N
N
Institut du Cancer de Montréal, Hôpital Notre-Dame, Sherbrooke, Canada.
Massie
B
B
Richer
M
M
Paradis
H
H
Langelier
Y
Y
D000256:Adenoviridae / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics*
D011994:Recombinant Proteins; D012264:Ribonucleotide Reductases