T7 rna polymerase variants with enhanced thermostability

Abstract

The present invention provides improved variants of T7 RNA polymerase by introducing novel mutations which lead to improved thermostability of the enzyme. According to the invention, amino acid substitutions at the positions Val426, Ser633, Val650, Thr654, Ala702, Val795, and combinations thereof are advantageous.

1 - 15 . (canceled) 
     
     
         16 . A method for producing a variant polypeptide of T7 RNA polymerase (T7 variant), the T7 variant having (i) a DNA-dependent RNA polymerase activity, (ii) an enhanced thermostability compared to SEQ ID NO: 2 (wild-type reference), and (iii) a different composition of amino acids compared to the wild-type reference, the method comprising the steps of:
 (a) selecting an amino acid from the group consisting of Val426, Ser633, Val650, Thr654, Ala702, and Val795, numbered from the N-terminus of the wild-type reference;   (b) substituting the selected amino acid with a different amino acid to form a T7 variant, wherein the different amino acid is selected from the group consisting of:
 Leu, and Ile when the different amino acid substitutes for Val426 (Val426Leu, Val426Ile), 
 Val and Met when the different amino acid substitutes for Ser633 (Ser633Val, Ser633Met), 
 Leu when the different amino acid substitutes for Val650 (Val650Leu), 
 Leu when the different amino acid substitutes for Thr654 (Thr654Leu), 
 Val when the different amino acid substitutes for Ala702 (Ala702Val), and 
 Ile when the different amino acid substitutes for Val795 (Val795Ile), 
   wherein at least one amino acid of the wild-type reference and up to four amino acids at different positions are substituted; and   (c) expressing a nucleic acid molecule with a nucleotide sequence encoding the T7 variant of step (b) in an expression system, and isolating the expressed T7 variant from the expression system, thereby producing the T7 variant.   
     
     
         17 . A method for producing a nucleic acid molecule with a nucleotide sequence encoding a variant polypeptide of T7 RNA polymerase (T7 variant), the T7 variant having (i) a DNA-dependent RNA polymerase activity, (ii) an enhanced thermostability compared to SEQ ID NO: 2 (wild-type reference), and (iii) a different composition of amino acids compared to the wild-type reference, the method comprising the steps of:
 synthesizing at least one nucleic acid molecule encoding at least one variant polypeptide of T7 RNA polymerase;   wherein the T7 variant comprises a polypeptide of the wild-type reference in which at least one amino acid and up to four amino acids at different positions are substituted,   wherein a different amino acid substitutes for an amino acid selected from the group consisting of Val426, Ser633, Val650, Thr654, Ala702, and Val795, numbered from the N-terminus of the wild-type reference, and   wherein the different amino acid is selected from the group consisting of
 Leu, Ile and Phe when the different amino acid substitutes for Val426 (Val426Leu, Val426Ile, Val426Phe), 
 Val and Met when the different amino acid substitutes for Ser633 (Ser633Val, Ser633Met), 
 Leu when the different amino acid substitutes for Val650 (Val650Leu), 
 Leu when the different amino acid substitutes for Thr654 (Thr654Leu), 
 Val when the different amino acid substitutes for Ala702 (Ala702Val), and 
 Ile when the different amino acid substitutes for Val795 (Val795Ile). 
   
     
     
         18 . A nucleic acid molecule with a nucleotide sequence encoding a variant polypeptide of T7 RNA polymerase (T7 variant), the T7 variant having (i) a DNA-dependent RNA polymerase activity, (ii) an enhanced thermostability compared to SEQ ID NO: 2 (wild-type reference), and (iii) a different composition of amino acids compared to the wild-type reference, the nucleic acid being obtainable by the method of  claim 17 . 
     
     
         19 . The nucleic acid molecule according to  claim 18 , wherein the nucleotide sequence of the nucleic acid comprises a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, and SEQ ID NO: 47. 
     
     
         20 . The nucleic acid molecule according to  claim 18 , wherein the nucleic acid molecule is functionally linked to one or more nucleotide sequences capable of controlling transcription and/or translation to form an expression vector. 
     
     
         21 . The nucleic acid molecule according to  claim 20 , wherein the expression vector transforms a host organism.