Recombinant antibodies for cytoplasmic, nuclear and transmembrane proteins

描述（由申请人提供）：本授权中提出的实验的目的是产生重组抗体样蛋白（胞内抗体），其可以标记所有类型的特异性蛋白，包括细胞质和核蛋白以及跨膜蛋白上的细胞外表位。最近，我们已经显示了效用的胞内抗体的方法产生的探针，识别突触后蛋白Gephyrin和PSD 95。这些探针工作效率高，可用于观察生物体内的内源性蛋白质。然而，这些胞内抗体的设计限制了它们可以针对的靶标。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策略，用于产生胞内抗体，不对靶蛋白的类型提出任何要求。为了确定该策略的功效，我们将产生针对运动蛋白驱动蛋白1、转录因子AP-1和AMPA受体GluA 1的细胞外表位的胞内抗体。

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.