Mutants of desoxycytidine kinase having extended enzymatic activity

摘要

A process for evolving a protein (X) to modify its properties, is new. A process for evolving a protein (X) to modify its properties, comprises first preparing cells of genotype [protein Y*::protein X+] by transforming [protein Y*] cells with a gene for protein X (Y* indicates an inactive gene for Y). Y belongs to a class related to X, and with similar activity, with X and Y having at least the first three figures of the 4-digit EC code the same. The cells are auxotrophic for the product of the reaction of Y on its substrate in the culture medium. The cells are exposed to mutagen, cultured in medium containing the substrate for Y (the product of which is necessary for survival) and those cells in which X, modified by the mutagen, complements the defect in Y are selected. Independent claims are also included for: (1) a mutated protein X produced by the method and having a broader spectrum of activity then X itself; (2) a nucleic acid (I) encoding a mutated kinase of (1), i.e. human DCK (deoxycytidine kinase) 1 containing at least one of the mutations D133E and R104Q; (3) a vector containing (I); (4) a host cell containing the vector of (3); (5) non-therapeutic, in vivomutagenesis of a selected DNA sequence present in a cell; and (6) 3 Escherichia colistrains of genotype delta deo tdk containing a vector that encodes wild-type or mutant human DCK1.