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    Selection of host cells expressing protein at high levels
    1.
    发明申请
    Selection of host cells expressing protein at high levels 有权
    选择高水平表达蛋白质的宿主细胞

    公开(公告)号：US20110014655A1

    公开(公告)日：2011-01-20

    申请号：US12807133

    申请日：2010-08-26

    申请人： Arie P. Otte ,  Henricus J. M. van Blokland ,  Theodorus H. J. Kwaks ,  Richard G. A. B. Sewalt

    发明人： Arie P. Otte ,  Henricus J. M. van Blokland ,  Theodorus H. J. Kwaks ,  Richard G. A. B. Sewalt

    IPC分类号： C12P21/00 ,  C12N5/10

    CPC分类号： C07K16/00 ,  C07K14/00 ,  C07K16/30

    摘要： The invention provides a DNA molecule comprising a multicistronic transcription unit coding for i) a selectable marker polypeptide functional in a eukaryotic host cell, and for ii) a polypeptide of interest, the polypeptide of interest having a translation initiation sequence separate from that of the selectable marker polypeptide, characterized in that the coding sequence for the polypeptide of interest is downstream from the coding sequence for the selectable marker in the multicistronic transcription unit, and the nucleic acid sequence coding for the selectable marker polypeptide comprises a mutation that decreases the translation efficiency of the selectable marker in a eukaryotic host cell. The invention also provides methods for obtaining host cells expressing a polypeptide of interest, the host cells comprising the DNA molecules of the invention. The invention further provides the production of polypeptides of interest, comprising culturing host cells comprising the DNA molecules according to the invention.

    摘要翻译： 本发明提供了一种DNA分子，其包含编码i）在真核宿主细胞中有功能的选择性标记多肽，以及ii）感兴趣的多肽的多顺反子转录单位，所述感兴趣的多肽具有与可选择的不同的翻译起始序列 标记多肽，其特征在于感兴趣的多肽的编码序列在多顺反子转录单位中的选择性标记的编码序列的下游，并且编码选择标记多肽的核酸序列包含降低翻译效率的突变 在真核宿主细胞中的选择性标记。 本发明还提供了获得表达目的多肽的宿主细胞的方法，宿主细胞包含本发明的DNA分子。 本发明进一步提供感兴趣的多肽的生产，包括培养包含根据本发明的DNA分子的宿主细胞。

    Selection of host cells expressing protein at high levels
    2.
    发明申请
    Selection of host cells expressing protein at high levels 有权
    选择高水平表达蛋白质的宿主细胞

    公开(公告)号：US20080085537A1

    公开(公告)日：2008-04-10

    申请号：US11899505

    申请日：2007-09-06

    申请人： Arie Otte ,  Theodorus Kwaks ,  Henricus van Blokland ,  Richard Sewalt

    发明人： Arie Otte ,  Theodorus Kwaks ,  Henricus van Blokland ,  Richard Sewalt

    IPC分类号： C12P21/04 ,  C12N15/11 ,  C12N15/87 ,  C12N5/06

    CPC分类号： C12N15/85 ,  C12N2830/46 ,  C12N2840/203

    摘要： The invention provides a DNA molecule comprising a multicistronic transcription unit coding for i) a polypeptide of interest, and for ii) a selectable marker polypeptide functional in a eukaryotic host cell, wherein the polypeptide of interest has a translation initiation sequence separate from that of the selectable marker polypeptide, and wherein the coding sequence for the polypeptide of interest is upstream from the coding sequence for the selectable marker polypeptide in the multicistronic transcription unit, and wherein an internal ribosome entry site is present downstream from the coding sequence for the polypeptide of interest and upstream from the coding sequence for the selectable marker polypeptide, and wherein the nucleic acid sequence coding for the selectable marker polypeptide in the coding strand comprises a GTG or a TTG start codon. Also provided are methods for obtaining host cells expressing a polypeptide of interest, such host cells comprising DNA molecules of the invention. Further provided is the production of polypeptides of interest, comprising culturing host cells comprising the DNA molecules of the invention.

    摘要翻译： 本发明提供了一种DNA分子，其包含编码i）感兴趣多肽的多顺反子转录单位，以及ii）在真核宿主细胞中有功能的选择性标记多肽，其中所述目标多肽具有与 选择性标记多肽，并且其中感兴趣的多肽的编码序列在多顺反子转录单位中的选择性标记多肽的编码序列的上游，并且其中内部核糖体进入位点存在于感兴趣的多肽的编码序列的下游 并且编码选择性标记多肽的编码序列的上游，并且其中编码链中编码选择性标记多肽的核酸序列包含GTG或TTG起始密码子。 还提供了获得表达目的多肽的宿主细胞的方法，所述宿主细胞包含本发明的DNA分子。 进一步提供感兴趣的多肽的生产，包括培养包含本发明的DNA分子的宿主细胞。

    Means and methods for regulating gene expression
    9.
    发明申请
    Means and methods for regulating gene expression 审中-公开
    调控基因表达的方法和方法

    公开(公告)号：US20080206813A1

    公开(公告)日：2008-08-28

    申请号：US11978483

    申请日：2007-10-26

    申请人： Arie P. Otte ,  Arthur L. Kruckeberg ,  David P. E. Satijn

    发明人： Arie P. Otte ,  Arthur L. Kruckeberg ,  David P. E. Satijn

    IPC分类号： C12P21/00 ,  C12N5/06

    CPC分类号： C07K14/005 ,  C12N15/63 ,  C12N15/67 ,  C12N15/85 ,  C12N2710/10322 ,  C12N2800/108 ,  C12N2830/00 ,  C12N2830/006 ,  C12N2830/48 ,  C12N2830/85 ,  C12N2840/203 ,  C12P21/02

    摘要： Described are means and methods for regulating gene expression and production of proteinaceous molecules. Described are methods for producing a proteinaceous molecule in a cell comprising selecting a cell for its suitability for producing the proteinaceous molecule, providing a nucleic acid encoding the proteinaceous molecule with a nucleic acid comprising a STAR (STabilizing Anti-Repression) sequence, expressing the resulting nucleic acid in the cell and collecting the proteinaceous molecule. Providing at least one STAR sequence to a nucleic acid encoding a proteinaceous molecule enhances production (yield) of the proteinaceous molecule by the host cell, increases the proportion of host cells with acceptable expression levels, and/or increases the stability of a gene expression level.

    摘要翻译： 描述了调节蛋白质分子的基因表达和产生的方法和方法。 描述了在细胞中产生蛋白质分子的方法，包括选择细胞用于产生蛋白质分子的适合性，提供编码蛋白质分子的核酸与包含STAR（稳定抗抑制）序列的核酸，表达所得的 核酸，并收集蛋白质分子。 向编码蛋白质分子的核酸提供至少一个STAR序列增强宿主细胞的蛋白质分子的产生（产量），增加具有可接受的表达水平的宿主细胞的比例，和/或增加基因表达水平的稳定性 。

    Method for improving protein production
    10.
    发明授权
    Method for improving protein production 有权
    改善蛋白质生产的方法

    公开(公告)号：US07413896B2

    公开(公告)日：2008-08-19

    申请号：US11157193

    申请日：2005-06-20

    申请人： Arie Pieter Otte ,  Henricus Johannes Maria van Blokland

    发明人： Arie Pieter Otte ,  Henricus Johannes Maria van Blokland

    IPC分类号： C12N15/00 ,  C12N15/63 ,  C12P21/06 ,  C07H21/04 ,  A61K31/70

    CPC分类号： C12N15/67 ,  C12N15/63

    摘要： The present invention relates to the production of proteins in a cell or host cell. The invention uses a TRAnscription Pause (TRAP) sequence to enhance a protein expression characteristic of a protein expression unit. The TRAP sequence is thought to prevent, at least in part, formation of antisense RNA or to, at least in part, prevent transcription to enter the protein expression unit. In one embodiment, the invention provides a method for expression of at least one protein of interest in a cell comprising providing the cell with at least one protein expression unit that comprises a promoter functionally linked to an open reading frame encoding at least one protein of interest, characterized in that the protein expression unit further comprises at least one TRAP sequence and wherein the TRAP sequence is functionally located downstream of the open reading frame and at least in part prevents formation of antisense RNA. In another embodiment, the TRAP sequence is functionally located upstream of the promoter and at least in part prevents transcription to enter the expression unit. Preferably, the expression protein unit further comprises at least one STabilizing Anti-Repressor sequence.

    摘要翻译： 本发明涉及在细胞或宿主细胞中产生蛋白质。 本发明使用TRAnscription暂停（TRAP）暂停（TRAP）序列来增强蛋白质表达单元的蛋白质表达特征。 认为TRAP序列至少部分地防止反义RNA的形成或至少部分地防止转录进入蛋白质表达单元。 在一个实施方案中，本发明提供了用于在细胞中表达至少一种目的蛋白质的方法，包括向细胞提供至少一种蛋白质表达单位，所述蛋白质表达单元包含与编码至少一种感兴趣的蛋白质的开放阅读框功能连接的启动子 其特征在于，所述蛋白质表达单元还包含至少一个TRAP序列，并且其中所述TRAP序列在功能上位于所述开放阅读框的下游，并且至少部分地防止反义RNA的形成。 在另一个实施方案中，TRAP序列在功能上位于启动子的上游，并且至少部分地防止转录进入表达单元。 优选地，所述表达蛋白单元还包含至少一种稳定抗阻抑物序列。