Production of the R2 subunit of ribonucleotide reductase from herpes simplex virus with prokaryotic and eukaryotic expression systems: higher activity of R2 produced by eukaryotic cells related to higher iron-binding capacity.
The R2 subunit of ribonucleotide reductase from herpes simplex virus type 2 was overproduced with prokaryotic and eukaryotic expression systems. The recombinant R2 purified by a two-step procedure exhibited a 3-fold higher activity when produced in eukaryotic cells. Precise quantification of the R2 concentration at each step of the purification indicated that the activity was not altered during the purification procedure. Moreover, we have observed that the level of R2 expression, in eukaryotic cells as well as in prokaryotic cells, did not influence R2 activity. Extensive characterization of the recombinant R2 purified from eukaryotic and prokaryotic expression systems has shown that both types of pure R2 preparations were similar in their 76 kDa dimer contents (more than 95%) and in their ability to bind the R1 subunit. However, we have found that the higher activity of R2 produced in eukaryotic cells is more probably related to a higher capability of binding the iron cofactor as well as a 3-fold greater ability to generate the tyrosyl free radical.
Lamarche
N
N
Institut du cancer de Montréal, Hôpital Notre-Dame, Canada.
Matton
G
G
Massie
B
B
Fontecave
M
M
Atta
M
M
Dumas
F
F
Gaudreau
P
P
Langelier
Y
Y
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D018258:Herpesvirus 2, Human / Q000201:enzymology*; D007501:Iron / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000235:genetics* / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D011994:Recombinant Proteins; D014443:Tyrosine; D007501:Iron; D012264:Ribonucleotide Reductases