Pharmaceutical Biotechnology, Eberhard Karls University Tuebingen, Tuebingen, Germany.
Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany.
Methods Mol Biol. 2022;2446:555-579. doi: 10.1007/978-1-0716-2075-5_29.
Single-domain antibodies such as nanobodies (Nbs) have substantially expanded the possibilities of advanced cellular imaging. In comparison to conventional antibodies, Nbs are characterized by small size, high stability, and solubility in many environments, including the cytoplasm. Nbs can be efficiently functionalized or modified according to the needs of the imaging approach. Target-specific Nbs can be easily converted into genetically encoded fluorescently labeled intrabodies, also known as chromobodies (CBs), which represent powerful tools to study the dynamics of different proteins of interest within living cells. In this context, CBs specific for a short peptide epitope provide a versatile alternative to bypass the limitations observed with larger fluorescent protein fusions and can be readily used to visualize and monitor peptide-tagged proteins for which specific Nbs are not available. Here, we present our novel detection system comprising a 15 amino acid peptide-tag (PepTag) in combination with a peptide-tag specific CB (PepCB). We provide protocols for adding the PepTag to different proteins of interest, reformatting the peptide-specific Nb (PepNb) into a CB for expression in mammalian cells, and establishment of stable cell lines expressing the PepCB for protein interaction assays and compound screenings.
单域抗体，如纳米抗体（Nbs），大大扩展了高级细胞成像的可能性。与传统抗体相比，Nbs 的特点是体积小、稳定性高、在许多环境中（包括细胞质）都具有溶解性。可以根据成像方法的需要，对 Nbs 进行有效的功能化或修饰。针对特定靶标的 Nbs 可以很容易地转化为遗传编码的荧光标记的内抗体，也称为色体（CBs），这是研究活细胞内不同感兴趣的蛋白质动力学的强大工具。在这种情况下，针对短肽表位的 CB 提供了一种通用的替代方法，可以绕过与较大荧光蛋白融合观察到的限制，并且可以很容易地用于可视化和监测没有特异性 Nb 的肽标记的蛋白质。在这里，我们提出了一个包含 15 个氨基酸肽标签（PepTag）的新型检测系统，结合了一个肽标签特异性 CB（PepCB）。我们提供了将 PepTag 添加到不同感兴趣的蛋白质、将肽特异性 Nb（PepNb）重新构建为在哺乳动物细胞中表达的 CB，以及建立表达 PepCB 的稳定细胞系用于蛋白质相互作用测定和化合物筛选的方案。
