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    Screening and enrichment system for protein expression in eukaryotic cells using a tricistronic expression cassette
    2.
    发明公开
    Screening and enrichment system for protein expression in eukaryotic cells using a tricistronic expression cassette 有权
    检查和使用的表达盒trizistronischen用于在真核细胞中表达蛋白质富集系统

    公开(公告)号：EP2700713A1

    公开(公告)日：2014-02-26

    申请号：EP12181138.4

    申请日：2012-08-21

    申请人： Miltenyi Biotec GmbH

    发明人： Schüler, Anne ,  Eckardt, Nicole Susann, Dr. ,  Bürger, Iris

    IPC分类号： C12N15/79 ,  C12N15/10

    CPC分类号： C12N15/65 ,  C12N15/52 ,  C12N2830/20 ,  C12N2840/203 ,  C12P21/00 ,  C12P21/02 ,  C12Q1/6897 ,  C12Q2563/107 ,  C12Q2565/626

    摘要： The present invention provides a polynucleotide and a method for screening and enriching a recombinant protein expressed in a eukaryotic cell, the polynucleotide comprising a tricistronic expression cassette comprising a) a promoter, b) a gene of interest (GOI), c) a reporter gene, d) a selection marker gene, e) an internal ribosome entry site (IRES) element, and f) a 2A element. Said method comprises a) transfecting or transducing suitable eukaryotic host cells with the polynucleotides of the present invention, b) culturing the eukaryotic host cells under conditions so as to express the protein of interest, the reporter protein and the selection marker protein in a cell culture selection medium suitable for selecting positively-transfected/transduced cells by means of said selection marker, and c) screening, sorting and/or enriching the cells expressing high levels of protein of interest by means of the reporter protein.

    摘要翻译： 本发明提供的多核苷酸和用于筛选和富集在真核细胞中过表达重组蛋白，所述多核苷酸包含一个三顺反子表达盒，其包含a）启动子，b）中的感兴趣的基因（GOI），c）中的报告基因的方法 ，D）的选择标记基因，E）内部核糖体进入位点（IRES）元件的元件，和f）2A。 所述方法包括：a）转染或转导合适的真核宿主细胞与本发明的多核苷酸，b）培养所述真核宿主细胞的条件下，以表达目的在细胞培养物的蛋白，报告蛋白和选择标记蛋白 选择培养基适于选择正转染/转导的细胞由所述选择标记物的手段;和c）筛选，分选和/或富集的细胞中，通过报道蛋白的手段表达高水平感兴趣的蛋白质的。