Cloning, Expression and Purification of a Mutant Tomato Thymidine Kinase 1 for Crystallography

Thymidine kinase 1 from tomato (ToTK1) is a newly discovered deoxyribonucleoside kinase (dNK) that has been subject to study due to its in vitro and in vivo properties making it an interesting suicide gene candidate for the treatment of brain tumors. The ToTK1 phosphorylates the nucleoside analogue 3-azido-2,3-dideoxythymidine (azidothymidine, AZT) equally well as its natural substrate thymidine (dThd). A truncated ToTK1 showed improved activity twice as high as the wild-type ToTK1, and also an increased specificity towards AZT. The combination of ToTK1 and AZT has been previously tested in two animal studies for its efficiency and use in suicide gene therapy for malignant glioma. In the present study, the C-terminal truncated version of ToTK1 (ToTK1ΔC25) has been successfully expressed in E. coli and purified by means of affinity chromatography (IMAC). Although, the purified ToTK1ΔC25 of bacterial origin displayed no significant enzymatic activity, expression was attempted in two insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (Hi5) using the baculovirus expression vector system (BEVS). Significant TK activities were detected in the crude extracts of insect cells transfected with recombinant baculoviruses bearing N- and C-terminal His-tagged ToTK1ΔC25 constructs. The findings of this project pave the way to determine the 3D structure of ToTK1 for understanding the structure-function relationships of nucleoside activation by this enzyme and thereby show routes towards further improvement of ToTK1 and other TK1-like dNKs for use in suicide gene therapy.Deoxyribonucleoside kinases (dNKs) are important enzymes in the synthesis of dNTPs, the building blocks of DNA, and also have several important applications in medicine and biotechnology. The thymidine kinase 1 from tomato (ToTK1) is a newly discovered dNK that has been subject to study due to its in vitro and in vivo properties. The ToTK1 has the ability to phosphorylate the nucleoside analogue 3-azido-2,3-dideoxythymidine (azidothymidine, AZT) equally well as its natural substrate thymidine (dThd). AZT has a safe clinical profile and can easily cross the blood brain barrier, which makes ToTK1/AZT a promising suicide gene/prodrug combination in the treatment of malignant glioma (brain tumors). The combination of ToTK1 and AZT has been previously tested in two animal studies and compared to the well-studied Herpes simplex virus thymidine kinase (HSV-TK) with ganciclovir as prodrug showing that ToTK1/AZT is a good alternative to HSV-TK/GCV. Furthermore, several mutants of ToTK1 with improved kinetic properties for AZT activation have been previously developed using random protein engineering and N- and C- terminal truncations. Interestingly, a truncated ToTK1 showed improved activity twice as high as the wild-type ToTK1, and also an increased specificity towards AZT. In the present study, the C-terminal truncated version of ToTK1 (ToTK1ΔC25) has been successfully expressed in E. coli and purified by means of affinity chromatography (IMAC). Although, the purified ToTK1ΔC25 of bacterial origin displayed no significant enzymatic activity, expression was attempted in two insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (Hi5) using the baculovirus expression vector system (BEVS). Significant TK activities were detected in the crude extracts of insect cells transfected with recombinant baculoviruses bearing N- and C-terminal His-tagged ToTK1ΔC25 constructs. The findings of this thesis pave the way to determine the 3D structure of ToTK1 for understanding the structure-function relationships of nucleoside activation by this enzyme and thereby show routes towards further rational improvements of ToTK1 and other TK1-like dNKs for use in suicide gene therapy