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1. 发明申请

US20130217067A1 Combinatorial DNA Library for Producing Modified N-Glycans In Lower Eukaryotes 审中-公开
标题翻译：用于在低等真核生物中生产修饰的N-糖的组合DNA文库
公开(公告)号：US20130217067A1
公开(公告)日：2013-08-22
申请号：US13408432
申请日：2012-02-29
申请人： TILLMAN U. GERNGROSS , Stefan Wildt , Byung-kwon Choi , Juergen Hermann Nett , Piotr Bobrowicz , Stephen R. Hamilton , Robert C. Davidson
发明人： TILLMAN U. GERNGROSS , Stefan Wildt , Byung-kwon Choi , Juergen Hermann Nett , Piotr Bobrowicz , Stephen R. Hamilton , Robert C. Davidson
IPC分类号： C07K14/47
CPC分类号： C12P21/005 , C07K14/47 , C12N9/1051 , C12N15/1082 , C12N15/79
摘要： The present invention relates to eukaryotic host cells having modified oligosaccharides which may be modified further by heterologous expression of a set of glycosyltransferases, sugar transporters and mannosidases to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. The invention provides nucleic acid molecules and combinatorial libraries which can be used to successfully target and express mammalian enzymatic activities such as those involved in glycosylation to intracellular compartments in a eukaryotic host cell. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation. Host cells with modified oligosaccharides are created or selected. N-glycans made in the engineered host cells have a Man5GlcNAc2 core structure which may then be modified further by heterologous expression of one or more enzymes, e.g., glycosyltransferases, sugar transporters and mannosidases, to yield human-like glycoproteins.
摘要翻译：本发明涉及具有修饰的寡糖的真核宿主细胞，其可以通过异源表达一组糖基转移酶，糖转运体和甘露糖苷酶进一步修饰，以成为用于产生哺乳动物例如人治疗性糖蛋白的宿主菌株。本发明提供核酸分子和组合文库，其可用于成功靶向和表达哺乳动物酶活性，例如参与糖基化的真核宿主细胞中的细胞内区室的那些。该方法提供了可用于表达和靶向参与糖基化的任何所需基因的工程化宿主细胞。建立或选择具有修饰寡糖的宿主细胞。在工程化宿主细胞中制备的N-聚糖具有Man5GlcNAc2核心结构，然后可以通过异源表达一种或多种酶，例如糖基转移酶，糖转运蛋白和甘露糖苷酶来进一步修饰，以产生人样糖蛋白。

2. 发明申请

US20120052530A1 Combinatorial DNA Library for Producing Modified N-Glycans in Lower Eukaryotes 审中-公开
标题翻译：用于在低等真核生物中生产修饰的N-糖的组合DNA文库
公开(公告)号：US20120052530A1
公开(公告)日：2012-03-01
申请号：US13156804
申请日：2011-06-09
申请人： TILLMAN U. GERNGROSS , Stefan Wildt , Byung-kwon Choi , Juergen Hermann Nett , Piotr Bobrowicz , Stephen R. Hamilton , Robert C. Davidson
发明人： TILLMAN U. GERNGROSS , Stefan Wildt , Byung-kwon Choi , Juergen Hermann Nett , Piotr Bobrowicz , Stephen R. Hamilton , Robert C. Davidson
IPC分类号： C12P21/00 , C12N1/19 , C40B40/08
CPC分类号： C12P21/005 , C07K14/47 , C12N9/1051 , C12N15/1082 , C12N15/79
摘要： The present invention relates to eukaryotic host cells having modified oligosaccharides which may be modified further by heterologous expression of a set of glycosyltransferases, sugar transporters and mannosidases to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. The invention provides nucleic acid molecules and combinatorial libraries which can be used to successfully target and express mammalian enzymatic activities such as those involved in glycosylation to intracellular compartments in a eukaryotic host cell. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation. Host cells with modified oligosaccharides are created or selected. N-glycans made in the engineered host cells have a Man5GlcNAc2 core structure which may then be modified further by heterologous expression of one or more enzymes, e.g., glycosyltransferases, sugar transporters and mannosidases, to yield human-like glycoproteins. For the production of therapeutic proteins, this method may be adapted to engineer cell lines in which any desired glycosylation structure may be obtained.
摘要翻译：本发明涉及具有修饰的寡糖的真核宿主细胞，其可以通过异源表达一组糖基转移酶，糖转运体和甘露糖苷酶进一步修饰，以成为用于产生哺乳动物例如人治疗性糖蛋白的宿主菌株。本发明提供核酸分子和组合文库，其可用于成功靶向和表达哺乳动物酶活性，例如参与糖基化的真核宿主细胞中的细胞内区室的那些。该方法提供了可用于表达和靶向参与糖基化的任何所需基因的工程化宿主细胞。建立或选择具有修饰寡糖的宿主细胞。在工程化宿主细胞中制备的N-聚糖具有Man5GlcNAc2核心结构，然后可以通过异源表达一种或多种酶，例如糖基转移酶，糖转运蛋白和甘露糖苷酶来进一步修饰，以产生人样糖蛋白。为了生产治疗性蛋白质，该方法可适用于工程化可能获得任何所需糖基化结构的细胞系。

3. 发明申请

US20120322100A1 METHODS FOR PRODUCING MODIFIED GLYCOPROTEINS 审中-公开
标题翻译：生产改性糖蛋白的方法
公开(公告)号：US20120322100A1
公开(公告)日：2012-12-20
申请号：US13461111
申请日：2012-05-01
申请人： TILLMAN U. GERNGROSS
发明人： TILLMAN U. GERNGROSS
IPC分类号： C12P21/00 , C12N1/19
CPC分类号： C12P21/005 , C07K2319/04 , C07K2319/05 , C12N1/14 , C12N9/1048 , C12N9/2488 , C12N15/79 , C12N15/80 , C12N15/81 , C12Y302/01 , C12Y302/01113
摘要： Cell lines having genetically modified glycosylation pathways that allow them to carry out a sequence of enzymatic reactions, which mimic the processing of glycoproteins I humans, have been developed. Recombinant proteins expressed in these engineered hosts yield glycoproteins more similar, if not substantially identical, to their human counterparts. The lower eukaryotes, which ordinarily produce high-mannose containing N-glycans such as Man5GlcNAC2 or other structures along human glycosylation pathways. This is achieved using a combination of engineering and/or selection of strains which: do not express certain enzymes which create the undesirable complex structures characteristic of the fungal glycoproteins, which express exogenous enzymes selected either to have optimal activity under the conditions present in the fungi where activity is desired, or which are targeted to an organelle where optimal activity is achieved, and combinations thereof wherein the genetically engineered eukaryote expresses multiple exogenous enzymes required to produce “human-like” glycoproteins.
摘要翻译：已经开发了具有遗传修饰的糖基化途径的细胞系，其允许它们进行模拟I型糖蛋白的加工的酶反应序列。在这些工程化宿主中表达的重组蛋白可以产生与其对应物更相似的糖蛋白（如果基本上不相同）。低等真核生物，其通常产生含有甘露糖的N-聚糖如Man5GlcNAC2或沿其人糖基化途径的其它结构。这是使用以下工程和/或选择的组合实现的：不表达产生真菌糖蛋白特征的不合需要的复合结构的某些酶，其表达选择在真菌中存在的条件下具有最佳活性的外源酶其中需要活性或靶向实现最佳活性的细胞器，以及其组合，其中遗传工程真核生物表达产生人样糖蛋白所需的多种外源酶。

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