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1. 发明公开

EP1785434A1 Targeting and tracing of antigens in living cells 审中-公开
标题翻译： Zenelen和Folgen von Antigenen在lebenden Zellen
公开(公告)号：EP1785434A1
公开(公告)日：2007-05-16
申请号：EP05024739.4
申请日：2005-11-11
申请人： Ludwig-Maximilians-Universität München
发明人： Rothbauer, Ulrich , Leonhardt, Heinrich
IPC分类号： C07K19/00 , C07K16/46
CPC分类号： G01N33/566 , C07K1/22 , C07K14/43595 , C07K16/18 , C07K2317/22 , C07K2317/92 , C07K2319/60
摘要： The present invention relates to a method of generating a detectable protein capable of binding an antigen of interest, comprising the steps of: (a) obtaining from an antibody producing cell of Camelidae or a pool of such cells, a first nucleic acid molecule or a pool of such nucleic acid molecules, encoding the variable region of an immunoglobulin or recombinantly or (semi)synthetically producing such first nucleic acid molecule or pool of first nucleic acid molecules; (b) optionally selecting from said pool a particular nucleic acid molecule encoding the variable region of a specific immunoglobulin; (c) fusing the coding region of the first nucleic acid molecule, encoding the variable region of an immunoglobulin in frame to the coding region of a second nucleic acid molecule, encoding a detectable marker (poly)peptide, wherein the coding region of the first nucleic acid molecule is located 5' of the coding region of the second nucleic acid molecule and wherein the coding regions are optionally separated by a coding region encoding a linker of at least one amino acid residue; and (d) expressing the fused nucleic acid molecule encoding the fusion protein in a cell or cell free extract. Furthermore, the present invention relates to a method for generating a library of detectable proteins capable of antigen binding and to a library generated by the methods of the present invention. Finally, the present invention also relates to a fusion protein comprising a first (poly)peptide sequence comprising the variable region of a heavy chain antibody of Camelidae and a second (poly)peptide sequence derivable from a fluorescent or chromophoric protein, wherein said (a) first (poly)peptide sequence is composed of framework 1, CDR1, framework 2, CDR2, framework 3 and CDR3, encoded by the nucleic acid sequence of SEQ ID NO: 2 or encoded by a nucleic acid sequence with at least 70% sequence identity or a fragment thereof; and (b) second (poly)peptide sequence is (i) the green fluorescent protein derivable from Aequorea victoria encoded by the nucleic acid sequence of SEQ ID NO: 7, or a fluorescent mutant or fragment thereof; (ii) the red fluorescent protein derivable from Discosoma (DsRed) encoded by the nucleic acid sequence of SEQ ID NO: 9, or a fluorescent mutant or fragment thereof; or (iii) a functional homologue of (i) or (ii) with at least 80% sequence identity, wherein said first (poly)peptide sequence is located N-terminally of said second (poly)peptide sequence, said sequences being optionally separated by a linker of at least one amino acid residues.
摘要翻译：本发明涉及产生能够结合感兴趣抗原的可检测蛋白质的方法，包括以下步骤：（a）从骆驼科的抗体产生细胞或这样的细胞池获得第一核酸分子或编码免疫球蛋白的可变区或重组或（半）合成地产生第一核酸分子或第一核酸分子的池的这种核酸分子的集合; （b）任选地从所述池选择编码特定免疫球蛋白的可变区的特定核酸分子; （c）将编码可检测标记（多）肽的编码可检测标记（多）肽的第二核酸分子的编码区编码框架中的免疫球蛋白的可变区的第一核酸分子的编码区融合，其中第一核酸分子位于第二核酸分子的编码区的5'，并且其中编码区任选地由编码至少一个氨基酸残基的接头的编码区分离; 和（d）在细胞或无细胞提取物中表达编码融合蛋白的融合核酸分子。此外，本发明涉及产生能够抗原结合的可检测蛋白质文库的方法和通过本发明的方法产生的文库。最后，本发明还涉及包含第一（多）肽序列的融合蛋白，所述第一（多）肽序列包含骆驼科的重链抗体的可变区和可衍生自荧光或发色蛋白的第二（多）肽序列，其中所述（）第一（多）肽序列由由SEQ ID NO：2的核酸序列编码或由具有至少70％序列的核酸序列编码的框架1，CDR1，框架2，CDR2，框架3和CDR3组成身份或其片段; 和（b）第二（多）肽序列是（i）可由SEQ ID NO：7的核酸序列编码的维他命水母的绿色荧光蛋白或荧光突变体或其片段; （ii）衍生自由SEQ ID NO：9的核酸序列编码的Discosoma（DsRed）的红色荧光蛋白或荧光突变体或其片段; 或（iii）具有至少80％序列同一性的（i）或（ii）的功能同系物，其中所述第一（多）肽序列位于所述第二（多）肽序列的N-末端，所述序列任选分离通过至少一个氨基酸残基的接头。

2. 发明授权

EP2055718B1 Targeting and tracing of antigens in living cells 有权转让
标题翻译：目标在活细胞抗原的调查和起诉
公开(公告)号：EP2055718B1
公开(公告)日：2012-12-26
申请号：EP08019358.4
申请日：2006-11-13
申请人： Ludwig-Maximilians-Universität München
发明人： Rothbauer, Ulrich , Leonhardt, Heinrich
IPC分类号： C07K16/18 , C07K16/46 , C07K19/00
CPC分类号： G01N33/566 , C07K1/22 , C07K14/43595 , C07K16/18 , C07K2317/22 , C07K2317/92 , C07K2319/60

3. 发明公开

EP2078750A1 A fluorescent two-hybrid (F2H) assay for direct visualization of protein interactions in living cells 有权
标题翻译： Fluoreszenter Zwei-Hybrid（F2H）-Assay zur direkten Sichtbarmachung von Proteininteraktionen in lebenden Zellen
公开(公告)号：EP2078750A1
公开(公告)日：2009-07-15
申请号：EP08000297.5
申请日：2008-01-09
申请人： Ludwig-Maximilians-Universität München
发明人： Rothbauer, Ulrich , Leonhardt, Heinrich , Zolghadr, Kourosh , Mortusewicz, Oliver
IPC分类号： C12N15/10 , G01N33/542 , C12Q1/68
CPC分类号： C12N15/1055 , C12N15/625 , G01N21/6486 , G01N33/542 , G01N33/6875 , G01N33/6884
摘要： The present invention relates to an in vitro method for detecting protein-protein interactions comprising: (a) expressing in a eukaryotic cell a first fusion protein comprising (i) a (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell or interacts with proteinaceous or non-proteinaceous structures accumulated at distinct sites in the nucleus of the cell; and (ii) a (poly)peptide specifically binding to GFP; (b) expressing in the same cell a second fusion protein comprising (i) GFP; and (ii) a bait (poly)peptide; (c) expressing in the same cell a third fusion protein comprising (i) a fluorescent (poly)peptide, the excitation and/or emission wavelength of which differs from that of GFP; and (ii) a prey (poly)peptide; and (d) detecting the fluorescence emission of the fluorescent parts of the second and the third fusion protein in the cell upon excitation, wherein a co-localization of the fluorescence emission of both fusion proteins in the cell nucleus is indicative of an interaction of the bait and the prey (poly)peptide. The invention also relates to an in vitro method for detecting protein-protein interactions comprising: (a) expressing in a eukaryotic cell a first fusion protein comprising (i) a fluorescent (poly)peptide; (ii) a (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell; and (iii) a bait (poly)peptide (b) expressing in the same cell a second fusion protein comprising (i) a fluorescent (poly)peptide, the excitation and/or emission wavelength of which differs from that of the fluorescent (poly)peptide comprised in said first fusion protein; and (ii) a prey (poly)peptide and (c) detecting the fluorescence emission of the fluorescent parts of the first and the second fusion protein in the cell upon excitation, wherein a co-localization of the fluorescence emission of both fusion proteins in the cell nucleus is indicative of an interaction of the bait and the prey (poly)peptide. Furthermore, the present invention relates to methods for identifying a compound modulating the interaction of two (poly)peptides and methods of determining the relative strength of the interaction of two proteins with a third protein.
摘要翻译：本发明涉及一种用于检测蛋白质 - 蛋白质相互作用的体外方法，包括：（a）在真核细胞中表达第一融合蛋白，所述第一融合蛋白包含（i）当在细胞中表达时在不同部位积累的（多）肽在细胞核中或与在细胞核中不同部位积聚的蛋白质或非蛋白质结构相互作用; 和（ii）与GFP特异性结合的（多）肽; （b）在相同细胞中表达第二融合蛋白，其包含（i）GFP; 和（ii）诱饵（多）肽; （c）在相同的细胞中表达第三融合蛋白，其包含（i）其激发和/或发射波长不同于GFP的荧光（多）肽; 和（ii）猎物（多）肽; 和（d）在激发时检测细胞中第二和第三融合蛋白的荧光部分的荧光发射，其中在细胞核中两种融合蛋白的荧光发射的共定位表明诱饵和猎物（多）肽。本发明还涉及用于检测蛋白质 - 蛋白质相互作用的体外方法，其包括：（a）在真核细胞中表达包含（i）荧光（多）肽的第一融合蛋白; （ii）当在细胞中表达时在细胞核中的不同部位积聚的（多）肽; 和（iii）在相同细胞中表达第二融合蛋白的诱饵（多）肽（b），其包含（i）荧光（多）肽，其激发和/或发射波长不同于荧光（聚）肽包含在所述第一融合蛋白中; 和（ii）猎物（多）肽和（c）在激发时检测细胞中第一和第二融合蛋白的荧光部分的荧光发射，其中两个融合蛋白的荧光发射的共定位细胞核指示诱饵和猎物（多）肽的相互作用。此外，本发明涉及用于鉴定调节两（多）肽的相互作用的化合物的方法以及确定两种蛋白质与第三种蛋白质相互作用的相对强度的方法。

4. 发明授权

EP2078750B1 A fluorescent two-hybrid (F2H) assay for direct visualization of protein interactions in living cells 有权
标题翻译：荧光双杂交（F2H）测定法用于在活细胞中的蛋白质相互作用的直接可视化
公开(公告)号：EP2078750B1
公开(公告)日：2016-09-28
申请号：EP08000297.5
申请日：2008-01-09
申请人： Ludwig-Maximilians-Universität München
发明人： Rothbauer, Ulrich , Leonhardt, Heinrich , Zolghadr, Kourosh , Mortusewicz, Oliver
IPC分类号： C12N15/10 , G01N33/542 , C12Q1/68
CPC分类号： C12N15/1055 , C12N15/625 , G01N21/6486 , G01N33/542 , G01N33/6875 , G01N33/6884

5. 发明公开

EP2055718A1 Targeting and tracing of antigens in living cells 有权转让
标题翻译： Zelelwertermittlung und Verfolgung von Antigenen在lebenden Zellen
公开(公告)号：EP2055718A1
公开(公告)日：2009-05-06
申请号：EP08019358.4
申请日：2006-11-13
申请人： Ludwig-Maximilians-Universität München
发明人： Rothbauer, Ulrich , Leonhardt, Heinrich
IPC分类号： C07K19/00 , C07K16/46
CPC分类号： G01N33/566 , C07K1/22 , C07K14/43595 , C07K16/18 , C07K2317/22 , C07K2317/92 , C07K2319/60
摘要： The present invention relates to a method of detecting the presence, amount or subcellular location of an antigenic structure of interest in a cell, comprising the steps of: (a) (i) expressing a fusion protein directed to the antigenic structure of interest in said cell or (ii) introducing a fusion protein directed to the antigenic structure of interest and coupled to a (poly)peptide capable of transducing into said cell; wherein said fusion protein comprises a first (poly)peptide sequence comprising the variable region of a heavy chain antibody of Camelidae and capable of specifically binding to a GFP epitope, and a second (poly)peptide sequence derivable from a fluorescent or chromophoric protein, wherein said (1.) first (poly)peptide sequence is composed of framework 1, CDR1, framework 2, CDR2, framework 3 and CDR3, encoded by the nucleic acid sequence of SEQ ID NO: 11 or 31 or encoded by a nucleic acid sequence with at least 70% sequence identity or a fragment thereof.
摘要翻译：本发明涉及一种检测感兴趣的抗原结构在细胞中的存在，数量或亚细胞位置的方法，包括以下步骤：（a）（i）（i）表达针对所述抗原结构的融合蛋白，引入针对感兴趣的抗原结构并与能够转导入所述细胞的（多）肽偶联的融合蛋白; 其中所述融合蛋白包含第一（多）肽序列，其包含Camelidae的重链抗体的可变区并且能够特异性结合GFP表位的第一（多）肽序列和可衍生自荧光或发色蛋白的第二（多）肽序列，其中所述（1.）第一（多）肽序列由由SEQ ID NO：11或31的核酸序列编码或由核酸序列编码的框架1，CDR1，框架2，CDR2，构架3和CDR3组成具有至少70％的序列同一性或其片段。

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