Mutants of deoxycytidine kinase having extended enzymatic activity

#The present invention relates to a process for artificial evolution in vivo of proteins, said process allowing a protein X to be evolved by complementation of a related protein Y, X and Y both belonging to the same class of enzyme commission (EC) nomenclature, or to related classes. The mutants D133E and R104Q of deoxycytidine kinase (DCK) were obtained, each of these mutations resulting in the acquisition of thymidine kinase activity by DCK.

#PCR sequencing and amplification techniques have recourse to an increasingly diversified range of nucleoside triphosphates, the activated monomers which can be condensed by DNA polmerases. Such artificial monomers are distinguished from the four natural monomers both by chemical alterations to the heterocyclic base [Sala et al., 1996] and to the pentose sugar and triphosphate group. Preparation of the triphosphate derivatives is generally carried out starting from the corresponding nucleosides by subjecting the free 5′ alcohol to phosphorylation, then by condensing the 5′ phosphate with pyrophosphate, in order to produce the triphosphate. Catalysis of the phosphorylation stage by a nucleoside kinase consuming ATP constitutes a process which is valid on the industrial scale. Such a synthesis process can only be envisaged with enzymes having an extended activity, i.e. nucleoside kinases capable of phosphorylating any nucleoside with similar effectiveness. More generally, obtaining enzymes having extended activities would make it possible to have access to powerful tools for any kind of biotechnical use.

#Various solutions for carrying out directed mutations in a DNA molecule have been described in the state of the art. These techniques consist of introducing in vitro a mutation, a deletion or an insertion into a specific site in a DNA molecule, for example by using PCR. These various techniques are described in Hall et al., Protein Eng. 4:601 (1991); Hemsley et al., Nucleic Acids Research 17:6545-6551 (1989); Ho et al., Gene 77:51-59 (1989); Hultman et al., Nucleic Acids Research 18:5107-5112 (1990); Jones et al., Nature 344:793-794 (1990); Jones et al., Biotechniques 12:528-533 (1992); Landt et al., Gene 96:125-128 (1990); Nassal et al., Nucleic Acids Research 18:3077-3078 (1990); Nelson et al., Analytical Biochemistry 180:147-151 (1989); Vallette et al., Nucleic Acids Research 17:723-733 (1989); Watkins et al., Biotechniques 15:700-704 (1993); Weiner et al., Gene 126:35-41 (1993); Yao et al., PCR Methods and Applications 1:205-207 (1992) and in Weiner et al., Gene 151:119-123 (1994). Besides the technical problems encountered, it is impossible to know in advance what would be the effect of a given mutation on the activity of a protein with such techniques.

#FIG. 1: Screen allowing the selection of a thymidine kinase activity in Escheria coli.

#FIG. 2: Compared specificities of the thymidine kinase of Escheria coli and human deoxycytidine kinase.

#FIG. 3: Examples of nucleoside analogues

#Chemical compounds: the compounds 3′-azido-3′dexoythymidine (AZT), 2′-deoxyinoside (dI), N4-amino-2′-deoxycitidine (2-hydroxy-4-hydrazino-pyrinidine deoxyribonucleoside, designated 4am′dC), 5-aza-2′-deoxycitidine (5azadC), 5-iodo-2′-deoxycytidine(5IdC), 5-bromo-2-deoxycytidine (5BrdC) and 5-methyl-2′-deoxycytidine (5MedC) were bought from Sigma. The 2-deoxy-isoadenosine (disoA) (2-amino-9-(2′-deoxy-β-D-ribofuranosyl)purine) was prepared by enzymatic transglycosylation using a crude extract of N-deoxyribosyltransferases of Lactobacillus leichmannii. Synthesis of 2′-deoxyisoguanosine (2-hydroxy-6-amino-9-(2′-deoxy-β-D-ribofuranosyl)purine) was carried out by closing the 5-amino-1-(2′-deoxy-β-D-ribofuranosyl)imidazole-4-carboxamide ring. The preparation of 1-(2′-deoxyribofuranosyl)imidazole-4-carboxamide (dY) has been described in Pochet et al, 1995.

#Culture of the bacterial strains: The bacteria were cultured in a rich Luria-Bertani (LB) medium or in a minimum medium supplemented with 2 g/l of glucose (MS). The same growth media were solidified with 15 g/l of agar (Difco) for the preparation of Petri dishes. The liquid and solid cultures were incubated at 37° C. In certain cases, antibiotics were added at the following concentrations: 100 mg/l of carbenicilline, 25 mg/l of kanamycin, 15 mg/l of tetracycline. Trimethoprim was used at a concentration of 100 mg/l in an LB medium supplemented with 0.3 mM of thymidine. For the induction of the gene expression, isopropyl-β-D-thiogalactoside (IPTG) was added at 0.5 mM.

#Strains and plasmid constructions: a list of E. coli K12 strains used and the plasmids constructed within the scope of the present invention is given in Table 1 below.