Highly purified recombinant varicella Zoster virus thymidine kinase is a homodimer.
Recombinant varicella zoster virus (VZV) thymidine kinase (TK) was isolated in a fast and gentle two-step procedure from Escherichia coli. The TK was expressed as a PreScission-cleavable fusion protein and purified by glutathione and ATP affinity chromatography, yielding homogeneous, highly pure VZV TK. The purified enzyme displays enzymatic activities with K(m) values of 0.3 +/- 0.06 microM for the natural substrate thymidine and 11.6 +/- 3.2 microM for ATP, indicating the biochemical equivalence with the viral VZV TK expressed in infected cells. Determinations of the native molecular weight by size exclusion chromatography and native polyacrylamide gel electrophoresis revealed that the pure enzyme is biologically active as a homodimer.
Amrhein
I
I
Department of Applied BioSciences, Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology, Winterthurerstrasse 190, Zürich, CH-8057, Switzerland.
Wurth
C
C
Bohner
T
T
Hofbauer
R
R
Folkers
G
G
Scapozza
L
L
D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005822:Genetic Vectors; D014645:Herpesvirus 3, Human / Q000737:chemistry*; D007700:Kinetics; D008970:Molecular Weight; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013937:Thymidine Kinase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D013937:Thymidine Kinase