PDF(Pubmed)
Abstract：
Cytoskeletal motor proteins are essential molecular machines that hydrolyze ATP to generate force and motion along cytoskeletal filaments. Members of the dynein and kinesin superfamilies play critical roles in transporting biological payloads (such as proteins, organelles, and vesicles) along microtubule pathways, cause the beating of flagella and cilia, and act within the mitotic and meiotic spindles to segregate replicated chromosomes to progeny cells. Understanding the underlying mechanisms and behaviors of motor proteins is critical to provide better strategies for the treatment of motor protein-related diseases. Here, we provide detailed protocols for the recombinant expression of the Kinesin-1 motor KIF5C using a baculovirus/insect cell system and provide updated protocols for performing single-molecule studies using total internal reflection fluorescence microscopy and optical tweezers to study the motility and force generation of the purified motor.
摘要：
细胞骨架运动蛋白是水解ATP以产生沿着细胞骨架细丝的力和运动的必要分子机器。动力蛋白和驱动蛋白超家族的成员在运输生物有效载荷（如蛋白质，细胞器,和囊泡)沿着微管途径，导致鞭毛和纤毛的跳动，并在有丝分裂和减数分裂纺锤体内作用，将复制的染色体分离到子代细胞。了解运动蛋白的潜在机制和行为对于为运动蛋白相关疾病的治疗提供更好的策略至关重要。这里,我们提供了使用杆状病毒/昆虫细胞系统重组表达Kinesin-1马达KIF5C的详细方案,并提供了使用全内反射荧光显微镜和光学镊子进行单分子研究的更新方案,以研究纯化马达的运动性和力的产生.
