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Novel deoxyribonucleside kinase enzyme multi-substrate variants

A deoxyribonucleoside kinase, enzyme variant technology, applied in the direction of enzymes, microorganisms, transferases, etc., can solve problems such as major by-products

Inactive Publication Date: 2003-07-09
沃尔夫冈・肯特 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] For example, ddNTPs used in sequencing as well as dNTPs used in PCR reactions are produced by chemical synthesis using toxic chemicals, resulting in a large number of by-products

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0254] PCR-induced Dm-dNK variants

[0255] A directed evolution approach based on mutagenesis PCR was used to generate mutant kinase forms. The open reading frame (ORF) of Dm-dNK was mutagenized by different nucleotide analog concentrations and the effect of different nucleotide analog concentrations was studied. The mutagenized PCR fragment was ligated into an expression plasmid and then transformed into a TK-deficient E. coli KY895 strain.

[0256] Random Mutagenesis and Mutation Library Construction

[0257] Expression vector pGEX-2T-rDm-dNK [Munch-Petersen et al. J. Biol. Chem. 2000 275(9) 6673-6679] was used as template for PCR mutagenesis.

[0258] The open reading frame (ORF) of Dm-dNK was amplified using the following primers:

[0259] Dm-TK3: 5'-CGCGGATCCATGGCGGAGGCAGCATCCT-3' (SEQ ID NO: 7); and

[0260] Dm-TK4: 5'-CGGAATTCTTATCTGGCGACCCTCTGGCGT-3' (SEQ ID NO: 8).

[0261] PCR is performed in two steps. PCR was first performed in a 20 μl reaction with 0.15...

Embodiment 2

[0273] Characterization of enzyme variants

[0274] sequencing

[0275] Using Thermo Sequenase radio-labelled terminator cycle sequencing kit and P 33 Purified plasmids were sequenced manually with tagged ddNTPs (Amersham Corp.) by the Sanger dideoxy method.

[0276] Determination of LD 100 (in vivo characterization)

[0277] To determine the lethal dose (LD) of nucleoside analogues 100 ) (at this lethal dose, the bacteria did not grow), logarithmic dilutions of the nucleoside analogs were performed and all clones showing increased sensitivity to at least one nucleoside analog were assayed on M9 plates. Plates with concentrations ranging from 10-1000 μM AraA, 3.16-1000 μM AraC; 0.01-100 μM AZT; 0.316-31.6 μM ddA; 0.0316-100 μM 2CdA or 10-3500 μM ddC were used to determine the LD of putative mutants 100(concentration capable of causing 100% death).

[0278] Plates were prepared by mixing media and the like at the lowest temperature possible prior to pouring the plat...

Embodiment 3

[0292] sequence determination

[0293] Publicly available expressed sequence tag (EST) libraries were searched with the basic local alignment search tool (BLAST) in the GenBank database of the National Institute for Biotechnology information to identify ESTs encoding enzymes similar to Dm-dNK (GenBanK ACCN AF226281) . Through this method, the ESTs ACCN AU004911 from Bombyx mori and ACCN AW159435 from Xenopus laevis were identified.

[0294] ESTs were obtained from Genome Research Group, National Institute of Radiological Sciences, Anagawa 4-9-1, Inage, Chiba 263-8555, Japan (ACCN AU004911) and Lita Annenberg Hazen Genome Sequencing Center, Cold Spring Harbor Laboratory, PO Box 100 , Cold Spring Harbor, NY 11724, USA (AW159435). The complete open reading frames of the deoxyribonucleoside kinases encoded by these two ESTs were determined by DNA sequencing (see Example 2).

[0295] The complete open reading frame was then submitted to GenBank and assigned numbers ACCNAF226281 ...

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Abstract

This invention relates to novel multi-substrate deoxyribonucleoside kinase variants. More specifically the invention provides novel deoxyribonucleoside kinase variants derived from insects or lower vertebrates, in particular from Drosophila melanogaster, from Bombyx mori, or from Xenopus laevis, novel polynucleotides encoding multi-substrate nucleoside kinase variants, vector constructs comprising the polynucleotide, host cells carrying the polynucleotide or vector, methods of sensitising cells to prodrugs, method of inhibiting pathogenic agents in warm-blooded animals, and pharmaceutical compositions comprising deoxyribonucleoside kinase variants of the invention.

Description

technical field [0001] The present invention relates to variants of novel multisubstrate deoxyribonucleoside kinases. More specifically, the present invention provides novel deoxyribonucleosides derived from insects or lower vertebrates, particularly from Drosophila melanogaster, from Bombyx mori, or from Xenopus laevis Kinase variants, novel polynucleotides encoding multi-substrate deoxyribonucleoside kinase variants, vector constructs containing the polynucleotides, host cells containing the polynucleotides or vectors, cells sensitized to prodrugs Methods, methods of inhibiting pathogens in warm-blooded animals, and pharmaceutical compositions comprising deoxyribonucleoside kinase variants of the invention. Background technique [0002] DNA is composed of 4 deoxyribonucleoside triphosphates supplied by de novo and salvage pathways. The key enzyme of this de novo pathway is ribonucleotide reductase, which catalyzes the reduction of the 2'-OH group of nucleoside diphosphat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K35/76A61K35/761A61K35/763A61K38/00A61K38/45A61K45/00A61K48/00A61P31/04A61P31/12A61P33/00A61P35/00A61P37/00C12N1/21C12N5/10C12N9/12
CPCC12N9/1205A61K38/00C12N2799/021A61P31/04A61P31/12A61P33/00A61P35/00A61P37/00
Inventor 沃尔夫冈·肯特比吉特·芒克-彼得森尤雷·皮斯克
Owner 沃尔夫冈・肯特
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