Recombinant antibodies for cytoplasmic, nuclear and transmembrane proteins

描述(申请人提供)：这项资助中提出的实验的目的是产生重组抗体样蛋白(体内)，可以标记所有类型的特定蛋白，包括细胞质和核蛋白，以及跨膜蛋白上的胞外表位。最近，我们通过产生识别突触后蛋白Gen和PSD95的探针，展示了体内方法的实用性。这些探针的工作效率很高，可以用来显示活体中的内源蛋白质。然而，这些体内的设计对它们可以针对的目标施加了限制。特别是，靶蛋白必须固定在细胞的细胞骨架上。在这里，我们建议开发一种新的生成体内的策略，该策略不对目标蛋白的类型提出任何要求。为了确定这一策略的有效性，我们将产生针对运动蛋白Kinesin 1、转录因子AP-1和AMPA受体GluA1的细胞外表位的体内抗体。

DESCRIPTION (provided by applicant): The purpose of the experiments proposed in this grant is to generate recombinant antibody-like proteins (intrabodies) that can label specific proteins of all types, including cytoplasmic and nuclear proteins, and extracellular epitopes on transmembrane proteins. Recently we have shown the utility of the intrabody approach by generating probes that recognize the postsynaptic proteins Gephyrin and PSD95. These probes work efficiently and can be used to visualize endogenous proteins in living organisms. However, the design of these intrabodies places constraints on the targets against which they can be made. In particular, target proteins must be fixed to the cytoskeleton of the cell. Here we propose to develop a new strategy for generating intrabodies that does not place any requirements on the type of target protein. To establish the efficacy of this strategy we will generate intrabodies against the motor protein Kinesin 1, the transcription factor AP-1 and an extracellular epitope of the AMPA receptor GluA1.