T7 rna polymerase variants with enhanced thermostability
AssigneeRoche Diagnostics Operations, Inc.
InventorsHarald Sobek, Johann-Peter Thalhofer, Rainer Mueller, Manfred Schmidt, Michael Greif, Armin Ruf, Christian Rudolph
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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.
Claims15
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,
- 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).
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.
Dependencies are inferred from the claim text.