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1.发明授权
公开(公告)号：US5223408A
公开(公告)日：1993-06-29
申请号：US728456
申请日：1991-07-11
IPC分类号： A61K38/00 , C07K14/705 , C12N9/72 , C12N15/10 , C40B40/02
CPC分类号： C40B40/02 , C07K14/705 , C12N15/102 , C12N15/1037 , C12N9/6459 , C12Y304/21069 , A61K38/00 , C07K2319/00 , C07K2319/033 , C07K2319/33
摘要： A screening method for the selection of mutagenized proteins that are normally secreted by cells is described. The method includes the development of a cloning vector for the expression of secretory proteins as fusion proteins on the cell surface of transfected mammalian cells. The secreted protein is displayed on the cell surface by fusion with the glycophospholipid membrane anchor of decay accelerating factor (DAF). Tissue-type plasminogen activator (t-PA), which is normally secreted, is used as a model protein. PCR mutagenesis is used to generate random mutations within the Kringle 1 (K1) domain of t-PA. Fluorescence activated cell sorting (FACS) is employed to screen for t-PA mutants possessing a loss of an epitope to a specific Mab, whose nonlinear binding domains overlap with the t-PA clearance receptor contact regions novel t-PA mutants designated N115S, N1425S, and K159R were discovered by this method.
摘要翻译： 描述了通常由细胞分泌的诱变蛋白质的筛选方法。 该方法包括开发用于在转染的哺乳动物细胞的细胞表面上表达分泌蛋白作为融合蛋白的克隆载体。 分泌的蛋白质通过与衰老加速因子（DAF）的糖磷脂膜锚点融合而显示在细胞表面上。 通常分泌的组织型纤溶酶原激活物（t-PA）用作模型蛋白。 PCR诱变用于在t-PA的Kringle 1（K1）结构域内产生随机突变。 荧光活化细胞分选（FACS）用于筛选具有特异性Mab的表位损失的t-PA突变体，其特异性Mab的非线性结合域与t-PA清除受体接触区重叠，称为N115S，N1425S的新型t-PA突变体 ，K159R。
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2.发明授权
公开(公告)号：US5736135A
公开(公告)日：1998-04-07
申请号：US389615
申请日：1995-02-13
IPC分类号： A61K38/00 , C07K14/705 , C12N9/72 , C12N15/10 , C40B40/02 , A61K38/49 , C12N9/48 , C12N9/64 , C12N15/55
CPC分类号： C40B40/02 , C07K14/705 , C12N15/102 , C12N15/1037 , C12N9/6459 , C12Y304/21069 , A61K38/00 , C07K2319/00 , C07K2319/033 , C07K2319/33
摘要： A screening method for the selection of mutagenized proteins that are normally secreted by cells is described. The method includes the development of a cloning vector for the expression of secretory proteins as fusion proteins on the cell surface of transfected mammalian cells. The secreted protein is displayed on the cell surface by fusion with the glycophospholipid membrane anchor of decay accelerating factor (DAF). Tissue-type plasminogen activator (t-PA), which is normally secreted, is used as a model protein. PCR mutagenesis is used to generate random mutations within the Kringle 1 (K1) domain of t-PA. Fluorescence activated cell sorting (FACS) is employed to screen for t-PA mutants possessing a loss of an epitope to a specific Mab, whose nonlinear binding domains overlap with the t-PA clearance receptor contact regions novel t-PA mutants designated N115S, N1425S, and K159R were discovered by this method.
摘要翻译： 描述了通常由细胞分泌的诱变蛋白质的筛选方法。 该方法包括开发用于在转染的哺乳动物细胞的细胞表面上表达分泌蛋白作为融合蛋白的克隆载体。 分泌的蛋白质通过与衰老加速因子（DAF）的糖磷脂膜锚点融合而显示在细胞表面上。 通常分泌的组织型纤溶酶原激活物（t-PA）用作模型蛋白。 PCR诱变用于在t-PA的Kringle 1（K1）结构域内产生随机突变。 荧光活化细胞分选（FACS）用于筛选具有特异性Mab的表位损失的t-PA突变体，其特异性Mab的非线性结合域与t-PA清除受体接触区重叠，称为N115S，N1425S的新型t-PA突变体 ，K159R。
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