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Cancers 2015, 7(2), 966-980; doi:10.3390/cancers7020819

New Variants of Tomato Thymidine Kinase 1 Selected for Increased Sensitivity of E. coli KY895 towards Azidothymidine

1
Department of Biology, Lund University, Lund 22362, Sweden
2
Lund Protein Production Platform, Lund University, Lund 22362, Sweden
3
Department of Science, Systems and Models, Roskilde University, Roskilde 4000, Denmark
We would like to dedicate this article to Prof. Jure Piškur, who passed away while this work was in progress.
*
Authors to whom correspondence should be addressed.
Academic Editor: Gabi Dachs
Received: 28 March 2015 / Revised: 19 May 2015 / Accepted: 27 May 2015 / Published: 8 June 2015
(This article belongs to the Special Issue Cancers Gene Therapy)
View Full-Text   |   Download PDF [561 KB, uploaded 8 June 2015]   |  

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. View Full-Text
Keywords: nucleosides; protein engineering; mutagenesis; suicide gene therapy; deoxynucleosides; deoxynucleotides; deoxynucleoside kinases; nucleoside analog; azidothymidine; prodrug activation nucleosides; protein engineering; mutagenesis; suicide gene therapy; deoxynucleosides; deoxynucleotides; deoxynucleoside kinases; nucleoside analog; azidothymidine; prodrug activation
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Christiansen, L.S.; Egeblad, L.; Munch-Petersen, B.; Piškur, J.; Knecht, W. New Variants of Tomato Thymidine Kinase 1 Selected for Increased Sensitivity of E. coli KY895 towards Azidothymidine. Cancers 2015, 7, 966-980.

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