公开(公告)号：US20170298405A1

公开(公告)日：2017-10-19

申请号：US15629040

申请日：2017-06-21

Applicant: Roche Diagnostics Operations, Inc.

Inventor： Harald Sobek ,  Michael Greif ,  Marco Thomann ,  Sebastian Malik

IPC: C12P21/00 ,  C12N9/10 ,  C07K16/00

CPC classification number: C12P21/005 ,  C07K16/00 ,  C07K2317/41 ,  C12N9/1081 ,  C12P19/44 ,  C12Y204/99001 ,  C12Y301/03

Abstract: The present disclosure is directed to the properties of certain glycosyltransferase variants having N-terminal truncation deletions or internal deletions. Any of the mutants disclosed in here exhibit α-2,6-sialyltransferase enzymatic activity in the presence of CMP-activated sialic acid as co-substrate, and in the presence of a suitable acceptor site. A fundamental finding documented in the present disclosure is that suchs enzyme are not only capable of catalyzing transfer of a sialidyl moiety but they are also capable of catalyzing hydrolytic cleavage of terminally bound sialic acid from a glycan.

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：US20160102333A1

公开(公告)日：2016-04-14

申请号：US14970734

申请日：2015-12-16

Applicant: Roche Diagnostics Operations, Inc.

Inventor： Tibor Czabany ,  Alfred Engel ,  Michael Greif ,  Christine Jung ,  Christiane Luley ,  Sebastian Malik ,  Rainer Mueller ,  Bernd Nidetzky ,  Doris Ribitsch ,  Katharina Schmoelzer ,  Helmut Schwab ,  Harald Sobek ,  Bernhard Suppmann ,  Marco Thomann ,  Sabine Zitzenbacher

IPC: C12P21/00 ,  C07K16/00 ,  C12N9/10

CPC classification number: C12P21/005 ,  C07K16/00 ,  C07K2317/14 ,  C07K2317/24 ,  C07K2317/41 ,  C12N9/1081 ,  C12P19/18 ,  C12Y204/99001

Abstract: The present disclosure is directed to the use of certain glycosyltransferase variants having N-terminal truncation deletions. It was found that the combination of two different truncation variants of human β-galactoside-α-2,6-sialyltransferase I (hST6Gal-I) exhibited different specific sialyltransferase enzymatic activities. In one example, under conditions wherein the first variant Δ89 hST6Gal-I catalyzed formation of bi-sialylated target molecules the second variant Δ108 hST6Gal-I catalyzed formation of mono-sialylated target molecules. Thus, disclosed are variants of mammalian glycosyltransferase, nucleic acids encoding the same, methods and means for recombinantly producing the variants of mammalian glycosyltransferase and use thereof, particularly for sialylating in a quantitatively controlled manner terminal acceptor groups of glycan moieties being part of glycoproteins such as immunoglobulins.

Abstract translation: 本公开涉及使用具有N-末端截短缺失的某些糖基转移酶变体。 发现人和半乳糖苷-α-2,6-唾液酸转移酶I（hST6Gal-1）的两种不同截短变体的组合表现出不同的特异性唾液酸转移酶酶活性。 在一个实例中，在第一变体和Dgr; 89hST6Gal-1催化双唾液酸化靶向分子的形成的条件下，第二变体Dgr; 108hST6Gal-1催化单唾液酸化靶分子的形成。 因此，公开了哺乳动物糖基转移酶的变体，编码它们的核酸，用于重组产生哺乳动物糖基转移酶变体的方法和手段及其用途，特别是以定量控制的方式唾液酸化作为糖蛋白部分的聚糖部分的末端受体基团，例如 免疫球蛋白。

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。