Deoxynucleoside kinase from insect cells for the synthesis of nucleoside monophosphates

Abstract

Recombinant kinase remaining stable during the synthesis of nucleoside monophosphate without the addition of stabilizing SH reagents, without stabilizing proteins and accepting all four natural deoxynucleotides, obtainable from insect cells such as e.g. Drosophila Melanogaster. In addition, the invention concerns DNA sequences, vectors, transformed cells, a method for production of the recombinant kinase as well as its use for preparing nucleoside monophosphates.

1 . Recombinant kinase remaining stable during the synthesis of nucleoside monophosphate without the addition of stabilizing SH reagents, without stabilizing proteins and accepting all four natural deoxynucleosides, obtainable from cells of nonvertebrate organisms.  
     
     
         2 . Recombinant kinase as claimed in  claim 1  obtainable from insect cells.  
     
     
         3 . Recombinant kinase as claimed in  claim 1  or  2  showing—in a purified form—a specific activity of at least 20 U/mg (1U=1 μmol/min) for all 4 natural deoxynucleosides.  
     
     
         4 . Recombinant kinase as claimed in one of the  claims 1  to  3  showing a specificity constant k c /K m  of >10000 M −1  s −1  for all natural deoxynucleosides.  
     
     
         5 . Recombinant kinase as claimed in one of the  claims 1  to  4  where the kinase has a half-life of t ½ ≧50 h in Tris buffer with 5 mM MgCl 2  and of t ½ ≧25 h in water at 37° C.  
     
     
         6 . Recombinant kinase as claimed in one of the  claims 1  to  5  showing a wide temperature optimum between 40 and 60° C.  
     
     
         7 . Recombinant kinase as claimed in one of the  claims 1  to  6 , obtainable from  Drosophila Melanogaster.    
     
     
         8 . DNA sequence encoding a kinase from  Drosophila Melanogaster  as claimed in one of the  claims 1  to  7 .  
     
     
         9 . DNA sequence as claimed in  claim 8  wherein primers with the sequences SEQ ID No. 2—8 hybridize onto this DNA sequence.  
     
     
         10 . Vector containing the DNA sequence as claimed in  claim 8  or  9  and a promoter.  
     
     
         11 . Host transformed with a vector as claimed in  claim 10 .  
     
     
         12 . Method for production of a recombinant kinase as claimed in one of the  claims 1  to  7 , wherein the following steps are performed: 
 1) isolation of the coding sequence of Dm-dNK,  
 2) cloning of the structure gene in preferred expression vectors for  E. coli  with inducible promoters,  
 3) transformation of the expression vectors in preferred  E. coli  host strains and  
 4) expression of the Dm-dNK gene in  E. coli  by appropriate induction.  
 
     
     
         13 . Use of a recombinant kinase obtainable from  Drosophila Melanogaster  as claimed in one of the  claims 1  to  7  for the synthesis of the nucleoside monophosphate.  
     
     
         14 . Method for production of a nucleoside monophosphate wherein a recombinant kinase as claimed in  claims 1  to  7  is used for the phosphorylation of a nucleoside.