title: New Variants of Tomato Thymidine Kinase 1 Selected for Increased Sensitivity of E. coli KY895 towards Azidothymidine.
abstract: Nucleoside analogues (NA) are prodrugs that are phosphorylated by deoxyribonucleoside kinases (dNKs) as the first step towards a compound toxic to the cell. During the last 20 years, research around dNKs has gone into new organisms other than mammals and viruses. Newly discovered dNKs have been tested as enzymes for suicide gene therapy. The tomato thymidine kinase 1 (ToTK1) is a dNK that has been selected for its in vitro kinetic properties and then successfully been tested in vivo for the treatment of malignant glioma. We present the selection of two improved variants of ToTK1 generated by random protein engineering for suicide gene therapy with the NA azidothymidine (AZT).We describe their selection, recombinant production and a subsequent kinetic and biochemical characterization. Their improved performance in killing of E. coli KY895 is accompanied by an increase in specificity for the NA AZT over the natural substrate thymidine as well as a decrease in inhibition by dTTP, the end product of the nucleoside salvage pathway for thymidine. The understanding of the enzymatic properties improving the variants efficacy is instrumental to further develop dNKs for use in suicide gene therapy.
authors.affiliation: Department of Biology, Lund University, Lund 22362, Sweden. Louise.Slot_Christiansen@biol.lu.se.|Lund Protein Production Platform, Lund University, Lund 22362, Sweden. Louise.Slot_Christiansen@biol.lu.se.
authors.forename: Louise Slot
authors.initials: LS
authors.lastname: Christiansen
authors.affiliation: Lund Protein Production Platform, Lund University, Lund 22362, Sweden. Louise.Egeblad@biol.lu.se.
authors.forename: Louise
authors.initials: L
authors.lastname: Egeblad
authors.affiliation: Department of Science, Systems and Models, Roskilde University, Roskilde 4000, Denmark. bmp@ruc.dk.
authors.forename: Birgitte
authors.initials: B
authors.lastname: Munch-Petersen
authors.affiliation: Department of Biology, Lund University, Lund 22362, Sweden.
authors.forename: Jure
authors.initials: J
authors.lastname: Piškur
authors.affiliation: Department of Biology, Lund University, Lund 22362, Sweden. Wolfgang.Knecht@biol.lu.se.|Lund Protein Production Platform, Lund University, Lund 22362, Sweden. Wolfgang.Knecht@biol.lu.se.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Knecht
keywords: azidothymidine; deoxynucleoside kinases; deoxynucleosides; deoxynucleotides; mutagenesis; nucleoside analog; nucleosides; prodrug activation; protein engineering; suicide gene therapy