Mutants of Deoxycytidine Kinase Having Extended Enzymatic Activity

Abstract

The invention relates to a method for artificial in vivo evolution of proteins, said method making it possible to bring about the evolution of a protein X by complementation of a relative protein Y, X and Y both belonging to the same class of enzyme commission (EC) nomenclature or belonging to related classes. The mutants D133E and R104Q of desoxycytidine kinase (DCK) were obtained; both of said mutations result in acquisition of thymidine kinase activity by DCK.

1 - 12 . (canceled) 
     
     
         13 . A kinase, which is a human DCK1 possessing the sequence filed in GENBANK under accession number M60527 comprising at least one mutation selected from the mutations D133E and R104Q. 
     
     
         14 - 30 . (canceled) 
     
     
         31 . The kinase of  claim 13 , wherein the kinase comprises the mutation D133E. 
     
     
         32 . The kinase of  claim 13 , wherein the kinase comprises the mutation R104Q. 
     
     
         33 . The kinase of  claim 13 , wherein the kinase comprises the mutation D133E and the mutation R104Q. 
     
     
         34 . Process for converting nucleoside analogues into their respective 5′ phosphate derivatives comprising contacting the nucleoside analogues with the kinase of  claim 13 . 
     
     
         35 . Process according to  claim 34 , wherein the nucleoside analogues are selected from ddA, ddI, araC, AZT, disoA, dl, 8ho′dl, d-oxanoside, amino-dC, and 5-aza-dC. 
     
     
         36 . Process for mutating a cell, wherein the process comprises:
 (a) cultivating the cell in a culture medium containing promutagenic nucleoside analogues;   (b) expressing the kinase of  claim 13  in the cell to convert the promutagenic nucleoside analogues into their respective 5′ phosphate derivatives; and   (c) incorporating the 5′ phosphate nucleoside derivatives obtained in step (b) into the cell's DNA by elongation.   
     
     
         37 . Process according to  claim 36 , wherein the promutagenic nucleoside analogues are further converted by an enzyme in order to extend the range of mutagenesis by means of promutagens. 
     
     
         38 . Process according to  claim 37 , wherein the enzyme is selected from adenosine monophosphate kinase (AMK) and transdeoxyribosylase (NID). 
     
     
         39 . Process according to  claim 36 , wherein the nucleoside analogues are selected from ddA, ddI, araC, AZT, disoA, dl, 8ho′dl, d-oxanoside, amino-dC, and 5-aza-dC.