Difference between revisions of "Part:BBa K404110"
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| + | Thymidine kinase (TK) (EC 2.7.1.21) is known to be involved in the salvage pathway of nucleosides to nucleotides (Andrei et al. 2005). | ||
| + | Due to its broader spectrum for different substrates, herpes simplex virus thymidine kinase (HSV-TK) is widely used in gene therapy approaches instead of endogenous thymidine kinases (Black et al. 1996). The transgenic introduced HSV-TK monophosphrylates nucleosides or nucleoside analogs such as ganciclovir (GCV) or acyclovir (AVC) followed by further phosphorylation through cellular kinases to nucleoside triphsphosphates. Incorporation of nucleotide analogs such as ganciclovir triphosphate or acyclovir triphosphates leads to DNA chain termination (Reardon 1989) and finally results in cell death. | ||
| + | Genetic modifications of the active site represented by a tripeptide motif in thymidine kinase increases the substrate affinity of HSV-TK towards GCV and ACV (Black et al. 1996). Two promising mutant HSV-TKs have been found by large mutagenesis screenings modifying several amino acids and conducting sensitivity assays for ganciclovir and acyclovir (Black et al. 2001). | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
| + | In contrast to the TK30 mutant, the modified thymidine kinase SR39 obtained from a semi-random (SR) mutagenesis screening contains five modifications listed in Table 1 and provides further specificity for nucleoside analogs ganciclovir and acyclovir. | ||
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| + | Table 1: Modified amino acid sequences of two mutant thymidine kinases. | ||
| + | Mutant Modified amino acid sequence | ||
| + | wt HSV-TK 152A 159L 160I 161F 168A 169L | ||
| + | TK 30 (Black et al. 1996) 152V 159I 160L 161A 168Y 169F | ||
| + | SR39 (Black et al. 2001) 152A 159I 160F 161L 168F 169M | ||
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Revision as of 19:59, 26 October 2010
Thyminidine kinase (mutant SR39)
Thymidine kinase (TK) (EC 2.7.1.21) is known to be involved in the salvage pathway of nucleosides to nucleotides (Andrei et al. 2005). Due to its broader spectrum for different substrates, herpes simplex virus thymidine kinase (HSV-TK) is widely used in gene therapy approaches instead of endogenous thymidine kinases (Black et al. 1996). The transgenic introduced HSV-TK monophosphrylates nucleosides or nucleoside analogs such as ganciclovir (GCV) or acyclovir (AVC) followed by further phosphorylation through cellular kinases to nucleoside triphsphosphates. Incorporation of nucleotide analogs such as ganciclovir triphosphate or acyclovir triphosphates leads to DNA chain termination (Reardon 1989) and finally results in cell death. Genetic modifications of the active site represented by a tripeptide motif in thymidine kinase increases the substrate affinity of HSV-TK towards GCV and ACV (Black et al. 1996). Two promising mutant HSV-TKs have been found by large mutagenesis screenings modifying several amino acids and conducting sensitivity assays for ganciclovir and acyclovir (Black et al. 2001).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 68
Illegal NgoMIV site found at 973 - 1000COMPATIBLE WITH RFC[1000]