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

    公开(公告)号：US20060195935A1

    公开(公告)日：2006-08-31

    申请号：US11416490

    申请日：2006-05-02

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

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

    IPC分类号： A01H1/00 ,  C07H21/04 ,  C12N15/82 ,  C12N5/04

    CPC分类号： C07K14/505 ,  C12N9/1205 ,  C12N15/67 ,  C12N15/85 ,  C12N2830/46 ,  C12N2840/20 ,  C12N2840/203 ,  C12N2840/206 ,  C12N2840/50 ,  C12Y207/01095

    摘要： The invention provides a DNA molecule comprising an open reading frame sequence that encodes a selectable marker polypeptide, wherein said DNA molecule in the coding strand comprises a translation start sequence for the selectable marker polypeptide having a GTG startcodon or a TTG startcodon, and wherein the open reading frame sequence that encodes the selectable marker protein has been mutated to replace at least half of its CpG dinucleotides as compared to the native open reading frame sequence that encodes the selectable marker protein. The invention further provides such DNA molecules wherein the open reading frame sequence that encodes a selectable marker polypeptide is part of a multicistronic transcription unit that further comprises an open reading frame sequence encoding a polypeptide of interest. The invention also provides methods for obtaining host cells expressing a polypeptide of interest, said 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： 本发明提供了包含编码选择性标记多肽的开放阅读框序列的DNA分子，其中编码链中的所述DNA分子包含具有GTG起始密码子或TTG起始密码子的可选择标记多肽的翻译起始序列，并且其中所述开放 与编码可选择标记蛋白的天然开放阅读框序列相比，编码可选择标记蛋白的阅读框序列已被突变以替代其至少一半的CpG二核苷酸。 本发明还提供了这样的DNA分子，其中编码可选择标记多肽的开放阅读框序列是多顺反子转录单位的一部分，其进一步包含编码目的多肽的开放阅读框序列。 本发明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。 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。

    Method for improving protein production
    3.
    发明申请
    Method for improving protein production 有权

    公开(公告)号：US20060010506A1

    公开(公告)日：2006-01-12

    申请号：US11157193

    申请日：2005-06-20

    申请人： Arie Otte ,  Henricus Blokland

    发明人： Arie Otte ,  Henricus Blokland

    IPC分类号： A01K67/027

    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.

    Selection of host cells expressing protein at high levels
    5.
    发明申请
    Selection of host cells expressing protein at high levels 审中-公开
    选择高水平表达蛋白质的宿主细胞

    公开(公告)号：US20060172382A1

    公开(公告)日：2006-08-03

    申请号：US11269525

    申请日：2005-11-07

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

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

    IPC分类号： C07H21/04 ,  C12P21/06 ,  C12N5/06 ,  C07K16/18

    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 said 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, said 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，其特征在于感兴趣多肽的编码序列在所述多顺反子转录单位中用于选择性标记的编码序列的下游，并且编码选择性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，所述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。

    Means and methods for producing a protein through chromatin openers that are capable of rendering chromatin more accessible to transcription factors
    6.
    发明申请
    Means and methods for producing a protein through chromatin openers that are capable of rendering chromatin more accessible to transcription factors 有权
    通过染色质开放剂生产蛋白质的方法和方法，其能使染色质更易于转录因子使用

    公开(公告)号：US20060003416A1

    公开(公告)日：2006-01-05

    申请号：US11156910

    申请日：2005-06-20

    申请人： Arie Otte ,  Theodorus Kwaks ,  Richard Bernardus Sewalt

    发明人： Arie Otte ,  Theodorus Kwaks ,  Richard Bernardus Sewalt

    IPC分类号： C12Q1/68 ,  C12P21/06 ,  C12N9/10 ,  C12N15/85

    CPC分类号： C12N15/635 ,  C12N15/63 ,  C12N15/85 ,  C12N2830/48 ,  C12N2840/203 ,  C12N2840/206

    摘要： Described are means and methods for providing a cell with a protein expression unit, the method comprising providing a nucleic acid sequence comprising the unit with a nucleic acid sequence encoding a binding site for a member of a chromatin modification system for rendering chromatin more accessible for transcription (opener), wherein the opener is present in the cell. Preferred openers comprise histone modification proteins, chromatin remodeling proteins and trithorax group proteins or equivalents. The cells thus generated and nucleic acid sequences encoding such openers are provided. Openers are preferred in the context of STAR and TRAP sequences.

    摘要翻译： 描述了用于向细胞提供蛋白质表达单元的方法和方法，所述方法包括提供包含该单元的核酸序列，该核酸序列编码编码染色质修饰系统成员的结合位点的核酸序列，以使得染色质更易于转录 （开环剂），其中开启剂存在于电池中。 优选的开放剂包括组蛋白修饰蛋白，染色质重塑蛋白和trithorax组蛋白或等同物。 提供由此产生的细胞和编码这种开放子的核酸序列。 在STAR和TRAP序列的背景下，开放者是首选的。

    Method for simultaneous production of multiple proteins; vectors and cells for use therein
    7.
    发明申请
    Method for simultaneous production of multiple proteins; vectors and cells for use therein 有权
    同时生产多种蛋白质的方法 载体和细胞用于其中

    公开(公告)号：US20050191723A1

    公开(公告)日：2005-09-01

    申请号：US11013031

    申请日：2004-12-14

    申请人： Arie Otte ,  Arthur Kruckeberg ,  Richard George Sewalt

    发明人： Arie Otte ,  Arthur Kruckeberg ,  Richard George Sewalt

    IPC分类号： C12N15/09 ,  C07H21/04 ,  C07K16/00 ,  C07K16/18 ,  C12N1/15 ,  C12N1/19 ,  C12N1/21 ,  C12N5/10 ,  C12N15/11 ,  C12N15/67 ,  C12N15/79 ,  C12N15/82 ,  C12N15/85 ,  C12N15/90 ,  C12Q1/02

    CPC分类号： C07K16/00 ,  C07K16/18 ,  C12N15/67 ,  C12N15/8218 ,  C12N15/822 ,  C12N15/8257

    摘要： The invention relates to the field of biochemistry, molecular biology, pharmacology and diagnosis. More specifically the present invention relates to the production of proteins in a host cell. Even more specifically, the invention relates to a method for improving expression of two or more proteins in a (host) cell. The method is suited for production of, for example, recombinant antibodies that can be used in a pharmaceutical preparation or as a diagnostic tool. In one embodiment, the invention provides a method for obtaining a cell which expresses two or more proteins comprising providing the cell with two or more protein expression units encoding two or more proteins, characterized in that at least two of the protein expression units comprise at least one STAR sequence.

    摘要翻译： 本发明涉及生物化学，分子生物学，药理学和诊断领域。 更具体地，本发明涉及在宿主细胞中产生蛋白质。 更具体地说，本发明涉及一种改善（宿主）细胞中两种或多种蛋白质表达的方法。 该方法适用于制备例如可用于药物制剂或作为诊断工具的重组抗体。 在一个实施方案中，本发明提供了获得表达两种或更多种蛋白质的细胞的方法，包括向细胞提供编码两种或更多种蛋白质的两种或更多种蛋白质表达单位，其特征在于至少两种蛋白质表达单元至少包含 一个STAR序列。

    Method of detecting a DNA sequence, a DNA sequence, a method of making a DNA construct and the use thereof
    8.
    发明申请
    Method of detecting a DNA sequence, a DNA sequence, a method of making a DNA construct and the use thereof 有权
    检测DNA序列的方法，DNA序列，DNA构建体的制造方法及其用途

    公开(公告)号：US20050106609A1

    公开(公告)日：2005-05-19

    申请号：US10972684

    申请日：2004-10-25

    申请人： Arie Otte

    发明人： Arie Otte

    IPC分类号： C12N15/09 ,  C12Q1/02 ,  C12Q1/68 ,  C12N15/74 ,  C12N15/85

    CPC分类号： C12Q1/6897

    摘要： The invention relates to a method of detecting a DNA sequence which at least partially contributes to promote the stable expression of a gene. To this end the DNA fragment to be examined is cloned in a vector between i) a DNA sequence involved in the induction of gene transcription repressing chromatin, and ii) a reporter gene. The invention also relates to a DNA sequence to be detected by means of the invention, and the application of a stable expression-enhancing DNA sequence for the stable expression of a gene.

    摘要翻译： 本发明涉及一种检测至少部分有助于促进基因稳定表达的DNA序列的方法。 为此，将待检查的DNA片段克隆在载体中，i）涉及诱导基因转录抑制染色质的DNA序列，和ii）报告基因。 本发明还涉及通过本发明检测的DNA序列和用于稳定表达基因的稳定表达增强DNA序列的应用。

    Selection of host cells expressing protein at high levels
    9.
    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。