T7 rna polymerase variants with cysteine-serine substitutions

Patent metadata

Field	Value
Publication number	US-2016010069-A1
Application number	US-201514872392-A
Country	US
Kind code	A1
Filing date	Oct 1, 2015
Priority date	Apr 1, 2011
Publication date	Jan 14, 2016
Grant date	—

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.

First claim

What is claimed is:

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 comprising an amino acid sequence of SEQ ID NO.:2, including a Cysteine residue at amino acid position between 510 and 530, numbered from the N-terminus of SEQ ID NO.:2, and a Serine residue substitution for the Cysteine residue at amino acid position 723, numbered from the N-terminus of SEQ ID NO.:2, 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 variant further 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 comprises a Cysteine-Serine substitution selected from the group consisting of Cys125Ser, Cys347Ser, Cys492Ser, Cys515Ser, and Cys839Ser.

4 . The aqueous solution according to claim 1 , wherein the variant further comprises an N-terminal His-tag.

5 . The aqueous solution according to claim 1 , wherein the variant further comprises an N-terminal Methionine.

6 . 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.

Assignees

• Roche Diagnostics Operations

Inventors

• Greif Michael
• Rudolph Christian
• Schmidt Manfred
• Sobek Harald
• Thalhofer Johann-Peter

Classifications

• C12N9/1247 Primary

  DNA-directed RNA polymerase (2.7.7.6) · CPC title

• C12Y207/07006

  DNA-directed RNA polymerase (2.7.7.6) · CPC title