Niwa Shinsuke, Chiba Kyoko
Frontier Research Institute for Interdisciplinary Sciences (FRIS), Tohoku University, Aramaki-Aoba 6-3, Aoba-Ku, Sendai, Miyagi, 980-0845, Japan.
Cytoskeleton (Hoboken). 2023 Sep-Oct;80(9-10):356-366. doi: 10.1002/cm.21756. Epub 2023 Apr 10.
Kinesin-1, a motor protein composed of the kinesin heavy chain (KHC) and the kinesin light chain (KLC), is essential for proper cellular morphogenesis and function. A monoclonal antibody (mAb) called H2 recognizes the KHC in a broad range of species and is one of the most widely used mAbs in cytoskeletal motor research. Here, we present vectors that express recombinant H2 in mammalian cells. We show the recombinant H2 performs as well as the hybridoma-derived H2 in both western blotting and immunofluorescence assays. Additionally, the recombinant H2 can detect all three human KHC isotypes (KIF5A, KIF5B, and KIF5C) and amyotrophic lateral sclerosis-associated KIF5A aggregates in cells. In addition, we developed a chickenized version of the H2 mAb's single chain variable fragment, which can be used in immunofluorescence microscopy and expands the potential applications of H2. Overall, our results demonstrate that recombinant H2 is a useful tool for studying the functions of KHCs.
驱动蛋白-1是一种由驱动蛋白重链（KHC）和驱动蛋白轻链（KLC）组成的运动蛋白，对细胞的正常形态发生和功能至关重要。一种名为H2的单克隆抗体（mAb）能在广泛的物种中识别KHC，是细胞骨架运动研究中使用最广泛的单克隆抗体之一。在此，我们展示了在哺乳动物细胞中表达重组H2的载体。我们发现重组H2在蛋白质免疫印迹和免疫荧光分析中表现与杂交瘤来源的H2一样好。此外，重组H2能检测细胞中的所有三种人类KHC同种型（KIF5A、KIF5B和KIF5C）以及与肌萎缩侧索硬化相关的KIF5A聚集体。另外，我们开发了H2单克隆抗体单链可变片段的鸡源化版本，其可用于免疫荧光显微镜检查，并扩展了H2的潜在应用。总体而言，我们的结果表明重组H2是研究KHC功能的有用工具。
