T7 rna polymerase variants with cysteine-serine substitutions

Abstract

The present disclosure provide novel variants of T7 RNA polymerase. Embodiments of T7 variants, according to the instant invention, include a Cysteine-Serine substitution on position 723 of the amino acid sequence of the T7 polypeptide. Embodiments of T7 variants according to the instant invention have a DNA-dependent RNA polymerase enzymatic activity and a reduced tendency to form intramolecular homodimers by way of oxidizing thiol groups. The amino acid substitutions within the T7 variants disclosed herein impact minimally, if at all, the RNA polymerase activity of the T7 polypeptide. Further, the mutations of the disclosed embodiments may optionally be combined with mutations which provide enhanced thermostability compared to the wild-type reference.

1 . An aqueous solution being devoid of a reducing agent with a thiol group, the aqueous solution comprising:
 a variant polypeptide of T7 RNA polymerase, the variant having DNA-dependent RNA polymerase activity and an amino acid sequence different from SEQ ID NO.:2,   wherein the variant includes a Cysteine residue at amino acid position between 510 and 530, numbered from the N-terminus of SEQ ID NO.:2,   wherein the variant includes a Serine residue substitution for the Cysteine residue at amino acid position 723, numbered from the N-terminus of SEQ ID NO.:2, and further wherein in the aqueous solution the variant is devoid of homomultimer formation of intermolecular disulfide bond(s).   
     
     
         2 . The aqueous solution according to  claim 1 , wherein the T7 variant comprises at least 2 and less than or equal to 10 amino acid substitutions as compared to SEQ ID NO.:2. 
     
     
         3 . The aqueous solution according to  claim 2 , wherein the variant further comprises a Cysteine-Serine substitution selected from the group consisting of Cys125Ser, Cys347Ser, Cys492Ser, Cys515Ser, and Cys839Ser. 
     
     
         4 . The aqueous solution according to  claim 2 , wherein the variant further comprises an amino acid substitution selected from the group consisting of Val426Leu, Val426Ile, Val426Phe, Ser633Val, Ser633Met, Val650Leu, Thr654Leu, Ala702Val, and Val795Ile. 
     
     
         5 . The aqueous solution according to  claim 4 , wherein the variant comprises amino acid substitutions Val426Leu, Val650Leu, Ala702Val, and Val795Ile. 
     
     
         6 . The aqueous solution according to  claim 4 , wherein the variant comprises amino acid substitutions Val426Leu, Ala702Val, and Val795Ile. 
     
     
         7 . The aqueous solution according to  claim 1 , further comprising an N-terminal His-tag linked to the variant. 
     
     
         8 . The aqueous solution according to  claim 1 , further comprising an N-terminal Methionine linked to the variant. 
     
     
         9 . The aqueous solution according to  claim 1 , wherein the reducing agent with the thiol group is selected from the group consisting of mercaptoethanol, dithiothreitol, dithioerythritol. 
     
     
         10 . A method of producing an aqueous solution devoid of a reducing agent including a thiol group, the solution comprising a variant of T7 RNA polymerase having DNA-dependent RNA polymerase activity and an amino acid sequence different from SEQ ID NO:2, the variant being devoid of homomultimer formation of intermolecular disulfide bonds when in the solution, the method comprising the steps of:
 providing the variant by substituting a Cysteine residue at amino acid position 723, numbered from the N-terminus of SEQ ID NO.:2, with a Serine residue;   reverse-transcribing the amino acid sequence of the variant, thereby obtaining a nucleotide sequence encoding the variant;   expressing a nucleic acid molecule comprising the nucleotide sequence of the variant obtained in the step of reverse transcribing in an expression system, thereby expressing a polypeptide; and   purifying the polypeptide expressed in the step of expressing, by way of chromatography using an aqueous mobile phase devoid of a reducing agent including a thiol group, thereby obtaining the solution with the variant.   
     
     
         11 . The method according to  claim 10 , wherein the solution obtained in the step of purifying is stored in the absence of a reducing agent with a thiol group. 
     
     
         12 . The method according to  claim 11 , wherein an amount of about 1 to 3 μg of the variant protein obtained in the step of purifying is detectably free of homomultimers, as determined by SDS polyacrylamide electrophoresis and staining with a Simply Blue Safe Stain Kit. 
     
     
         13 . The method according to  claim 10 , wherein the nucleic acid molecule comprising the nucleotide sequence of the variant comprises a T7 promoter functionally linked to a target nucleotide sequence to be transcribed. 
     
     
         14 . The method according to  claim 10 , wherein the expression system includes a buffer with one or more ribonucleoside triphosphates. 
     
     
         15 . The method according to  claim 10 , wherein the variant further comprises a Cysteine-Serine substitution selected from the group consisting of Cys125Ser, Cys347Ser, Cys492Ser, Cys515Ser, and Cys839Ser. 
     
     
         16 . The method according to  claim 10 , wherein the variant further comprises an amino acid substitution selected from the group consisting of Val426Leu, Val426Ile, Val426Phe, Ser633Val, Ser633Met, Val650Leu, Thr654Leu, Ala702Val, and Val795Ile. 
     
     
         17 . A method of synthesizing a RNA molecule, comprising the steps of:
 providing an aqueous solution devoid of a reducing agent with a thiol group, the solution having a variant polypeptide of T7 RNA polymerase, the variant having DNA-dependent RNA polymerase activity and an amino acid sequence different from SEQ ID NO:2, the variant being devoid of homomultimer formation of intermolecular disulfide bonds when in the solution;   providing a template DNA molecule comprising a T7 promoter functionally linked to a target nucleotide sequence to be transcribed;   contacting, within the solution, the template DNA molecule with the variant in the presence of ribonucleoside triphosphates; and   incubating the solution, following the step of contacting, under conditions favoring RNA polymerase activity, thereby synthesizing the RNA molecule.   
     
     
         18 . The method according to  claim 15 , wherein the variant further comprises a Cysteine-Serine substitution selected from the group consisting of Cys125Ser, Cys347Ser, Cys492Ser, Cys515Ser, and Cys839Ser. 
     
     
         19 . The method according to  claim 15 , wherein the variant further comprises an amino acid substitution selected from the group consisting of Val426Leu, Val426Ile, Val426Phe, Ser633Val, Ser633Met, Val650Leu, Thr654Leu, Ala702Val, and Val795Ile. 
     
     
         20 . The method according to  claim 15 , wherein the variant comprises amino acid substitutions Val426Leu, Ala702Val, and Val795Ile.