Thymidine Kinase Mutants

Abstract

The present invention provides isolated nucleic acid molecules encoding a Herpesviridae thymidine kinase enzyme comprising one or more mutations, at least one of the mutations encoding an amino acid substitution located toward the N-terminus from a DRH nucleoside binding site which increases a biological activity of the thymidine kinase, as compared to unmutated thymidine kinase. Such mutations include amino acid substitutions within a Q substrate binding domain which increases a biological activity of the thymidine kinase, as compared to unmutated thymidine kinase. Within a further aspect, fusion proteins are provided which have both guanylate kinase and thymidine kinase biological properties. Also provided are vectors suitable for expressing such DNA molecules, as well as methods for utilizing such vectors.

1 - 60 . (canceled) 
     
     
         61 . An isolated mutant Herpesviridae thymidine kinase of SEQ ID NO:134 with an amino acid substitution at one or more of positions 159-161, 168 and 169. 
     
     
         62 . The isolated thymidine kinase of  claim 61 , wherein said amino acid substitutions are at positions 160, 161, 168 and 169, and wherein said amino acid substitution at position 160 is isoleucine to phenylalanine, said amino acid substitution at position 161 is phenylalanine to leucine, said amino acid substitution at position 168 is alanine to phenylalanine, and said amino acid substitution at position 169 is leucine to asparagine. 
     
     
         63 . The isolated thymidine kinase of  claim 61 , wherein said amino acid substitutions are at positions 160, 161 and 168, and wherein said amino acid substitution at position 160 is isoleucine to phenylalanine, said amino acid substitution at position 161 is phenylalanine to alanine, and said amino acid substitution at position 168 is alanine to phenylalanine. 
     
     
         64 . The isolated thymidine kinase of  claim 61 , wherein said amino acid substitutions are at positions 159, 160, 161, 168 and 169, and wherein said amino acid substitution at position 159 is leucine to isoleucine, said amino acid substitution at position 160 is isoleucine to phenylalanine, said amino acid substitution at position 161 is phenylalanine to leucine, said amino acid substitution at position 168 is alanine to phenylalanine, and said amino acid substitution at position 169 is leucine to methionine. 
     
     
         65 . The isolated thymidine kinase of  claim 61 , wherein said amino acid substitutions are at positions 159, 160, 161, 168 and 169, and wherein said amino acid substitution at position 159 is leucine to isoleucine, said amino acid substitution at position 160 is isoleucine to phenylalanine, said amino acid substitution at position 161 is phenylalanine to alanine, said amino acid substitution at position 168 is alanine to tyrosine, and said amino acid substitution at position 169 is leucine to tyrosine. 
     
     
         66 . The isolated thymidine kinase of  claim 61 , wherein said amino acid substitutions are at positions 160, 161, 168 and 169, and wherein said amino acid substitution at position 160 is isoleucine to phenylalanine, said amino acid substitution at position 161 is phenylalanine to alanine, said amino acid substitution at position 168 is alanine to tyrosine, and said amino acid substitution at position 169 is leucine to tyrosine. 
     
     
         67 . The isolated thymidine kinase of  claim 61 , wherein said amino acid substitutions are at positions 160, 161, 168 and 169, and wherein said amino acid substitution at position 160 is isoleucine to phenylalanine, said amino acid substitution at position 161 is phenylalanine to valine, said amino acid substitution at position 168 is alanine to valine, and said amino acid substitution at position 169 is leucine to methionine. 
     
     
         68 . The isolated thymidine kinase of  claim 61 , wherein said amino acid substitutions are at positions 159, 160, 161, 168 and 169, and wherein said amino acid substitution at position 159 is leucine to isoleucine, said amino acid substitution at position 160 is isoleucine to phenylalanine, said amino acid substitution at position 161 is phenylalanine to valine, said amino acid substitution at position 168 is alanine to phenylalanine, and said amino acid substitution at position 169 is leucine to tyrosine.