公开(公告)号：US20160010069A1

公开(公告)日：2016-01-14

申请号：US14872392

申请日：2015-10-01

Applicant: Roche Diagnostics Operations, Inc.

Inventor： Michael Greif ,  Christian Rudolph ,  Manfred Schmidt ,  Harald Sobek ,  Johann-Peter Thalhofer

IPC: C12N9/12

CPC classification number: C12N9/1247 ,  C12Y207/07006

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.

Abstract translation: 本公开提供了T7RNA聚合酶的新变体。 根据本发明的T7变体的实施方案包括T7多肽的氨基酸序列的位置723上的半胱氨酸 - 丝氨酸取代。 根据本发明的T7变体的实施方案具有DNA依赖性RNA聚合酶酶活性，并且通过氧化硫醇基形成分子内同型二聚体的趋势降低。 本文公开的T7变体内的氨基酸取代最小程度地影响T7多肽的RNA聚合酶活性。 此外，所公开的实施方案的突变可以任选地与与野生型参考物相比提供增强的热稳定性的突变组合。

公开(公告)号：US09663769B2

公开(公告)日：2017-05-30

申请号：US14872392

申请日：2015-10-01

Applicant: Roche Diagnostics Operations, Inc.

Inventor： Michael Greif ,  Christian Rudolph ,  Manfred Schmidt ,  Harald Sobek ,  Johann-Peter Thalhofer

IPC: C12N9/12

CPC classification number: C12N9/1247 ,  C12Y207/07006

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.

公开(公告)号：US20160032261A1

公开(公告)日：2016-02-04

申请号：US14885732

申请日：2015-10-16

Applicant: ROCHE DIAGNOSTICS OPERATIONS, INC.

Inventor： Harald Sobek ,  Johann-Peter Thalhofer ,  Rainer Mueller ,  Manfred Schmidt ,  Michael Greif ,  Armin Ruf ,  Christian Rudolph

IPC: C12N9/12 ,  C12P19/34

CPC classification number: C12N9/1247 ,  C12P19/34 ,  C12Y207/07006

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.

Abstract translation: 本发明通过引入导致酶的热稳定性的新突变来提供T7RNA聚合酶的改进变体。 根据本发明，位置Val426，Ser633，Val650，Thr654，Ala702，Val795及其组合的氨基酸取代是有利的。

公开(公告)号：US20160032260A1

公开(公告)日：2016-02-04

申请号：US14885727

申请日：2015-10-16

Applicant: ROCHE DIAGNOSTICS OPERATIONS, INC.

Inventor： Harald Sobek ,  Johann-Peter Thalhofer ,  Rainer Mueller ,  Manfred Schmidt ,  Michael Greif ,  Armin Ruf ,  Christian Rudolph

IPC: C12N9/12

CPC classification number: C12N9/1247 ,  C12P19/34 ,  C12Y207/07006

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.