公开(公告)号：US20160102298A1

公开(公告)日：2016-04-14

申请号：US14970832

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: C12N9/10

CPC classification number: C12N9/1081 ,  C12N9/1048 ,  C12Y204/99001

Abstract: The present disclosure is directed to glycosyltransferase variants having N-terminal truncation deletions. Contrary to previous findings certain truncations comprising the conserved amino acid motif (“QVWxKDS”) were found to be compatible with glycosyltransferase enzymatic activity, particularly in a human sialyltransferase (hST6Gal-I). 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 terminal acceptor groups of glycan moieties being part of glycoproteins such as immunoglobulins.

Abstract translation: 本公开涉及具有N-末端截短缺失的糖基转移酶变体。 与之前的发现相反，发现包含保守氨基酸基序（“QVWxKDS”）的某些截短与糖基转移酶酶活性相容，特别是在人唾液酸转移酶（hST6Gal-1）中。 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，特别是用作糖蛋白的部分的聚糖末端受体基团，例如免疫球蛋白。

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.

公开(公告)号：US09809835B2

公开(公告)日：2017-11-07

申请号：US14950443

申请日：2015-11-24

Applicant: Roche Diagnostics Operations, Inc.

Inventor： Alfred Engel ,  Michael Greif ,  Christine Jung ,  Sebastian Malik ,  Rainer Mueller ,  Harald Sobek ,  Bernhard Suppmann ,  Marco Thomann

IPC: C12P19/44 ,  C12P21/00 ,  C12N9/10 ,  C12P19/02 ,  C12P19/18

CPC classification number: C12P19/18 ,  C12N9/1081 ,  C12P19/02 ,  C12P19/44 ,  C12P21/005

Abstract: The present disclosure is directed to the use of certain glycosyltransferase variants having N-terminal truncation deletions. Contrary to previous findings certain truncations were found to exhibit sialidase enzymatic activity, particularly a variant of human sialyltransferase (hST6Gal-I) with a truncation deletion involving the first 89 N-terminal amino acids of the respective wild-type polypeptide. A fundamental finding documented in the present disclosure is that there exists a variant of this enzyme which is capable of catalyzing transfer of a glycosyl moiety as well as hydrolysis thereof. Thus, disclosed is a specific exemplary variant of mammalian glycosyltransferase, nucleic acids encoding the same, methods and means for recombinantly producing the variant 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.

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。

公开(公告)号：US09481902B2

公开(公告)日：2016-11-01

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: C07K16/00 ,  C12Q1/00 ,  C12P21/00 ,  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β-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Δ89hST6Gal-1催化双唾液酸化靶分子的形成的条件下，第二变体Δ108hST6Gal-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：US20160076068A1

公开(公告)日：2016-03-17

申请号：US14950443

申请日：2015-11-24

Applicant: Roche Diagnostics Operations, Inc.

Inventor： Alfred Engel ,  Michael Greif ,  Christine Jung ,  Sebastian Malik ,  Rainer Mueller ,  Harald Sobek ,  Bernhard Suppmann ,  Marco Thomann

IPC: C12P19/18 ,  C12P19/02

CPC classification number: C12P19/18 ,  C12N9/1081 ,  C12P19/02 ,  C12P19/44 ,  C12P21/005

Abstract: The present disclosure is directed to the use of certain glycosyltransferase variants having N-terminal truncation deletions. Contrary to previous findings certain truncations were found to exhibit sialidase enzymatic activity, particularly a variant of human sialyltransferase (hST6Gal-I) with a truncation deletion involving the first 89 N-terminal amino acids of the respective wild-type polypeptide. A fundamental finding documented in the present disclosure is that there exists a variant of this enzyme which is capable of catalyzing transfer of a glycosyl moiety as well as hydrolysis thereof. Thus, disclosed is a specific exemplary variant of mammalian glycosyltransferase, nucleic acids encoding the same, methods and means for recombinantly producing the variant 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。 发现某些截短表现出唾液酸酶酶活性，特别是具有涉及相应野生型多肽的前89个N-末端氨基酸的截短缺失的人唾液酸转移酶（hST6Gal-1）的变体。 在本公开文献中记载的基本发现是存在能够催化糖基部分的转移以及其水解的酶的变体。 因此，公开了哺乳动物糖基转移酶的特定示例性变体，编码哺乳动物糖基转移酶的核酸，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部分的聚糖部分的末端受体基团 糖蛋白如免疫球蛋白。