Purification and Characterization of Herpes Simplex Virus ( Type 1 ) Thymidine Kinase Produced in Escherichia coli by a High Efficiency Expression Plasmid Utilizing a X PL Promoter and a cI 857 Temperature - sensitive Repressor
نویسنده
چکیده
The structural gene for herpes simplex virus (type 1) thymidine kinase was cloned downstream from the X phage high efficiency leftward promotor in a plasmid (pHETK2) also containing the gene for the X cI857 temperature-sensitive repressor. Thymidine kinase is synthesized as a run-on product containing the NH2 terminus of the X N protein. Heat inactivation of the X repressor by growth at 42 O C results in the accumulation of thymidine kinase as approximately 4% of the total soluble cellular protein. Thymidine kinase has been purified to greater than 95% homogeneity by high speed centrifugation, ammonium sulfate fractionation, and Sephadex G-100 and hydroxylapatite column chromatography. Thymidine kinase has a subunit M. = 42,000 determined by sodium dodecyl sulfate-polyacrylamide gel eleztrophoresis and behaves a a dimer during Sephadex G100 chromatography and glycerol gradient centrifugation. Thymidine kinase is enzymatically active from pH 6 to 10 with maximum activity at pH 8.5. The enzyme is protected from heat inactivation by thymidine and has a half-life at 40°C of 30 min in the presence of thymidine and 3 min in its absence. Thymidine kinase displays Michaelis-Menten kinetics with apparent Michaelis constants of 0.6 and 118 NM for thymidine and ATP, respectively. Iododeoxycytidine is a competitive inhibitor of thymidine with an apparent Ki of 14 HM. The anti-herpes drug acyclovir (9-[(2-hydroxyethoxy)methyl]guanine) also appears to be a cGmpetitive inhibitor of thymidine (Ki of approximately 300 PM) but requires 3,000-fold higher concentrations than thymidine to give 50% inhibition. Other nucleoside triphosphates can substitute for ATP in the kinase reaction with the exception of dTTP which appears to inhibit thymidine kinase activity by about 50% when present in concentrations equal to that of thymidine.
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