In vitro motility from recombinant dynein heavy chain
In vitro motility from recombinant dynein heavy chain
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DOI：
10.1073/pnas.93.13.6552
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发表时间：
1996-06-25
影响因子：
11.1
通讯作者：
Vallee, RB
中科院分区：
文献类型：
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作者：
Mazumdar, M;Mikami, A;Vallee, RB
动力蛋白是一类参与纤毛和鞭毛运动、细胞器运输和染色体分离的运动蛋白。由于它们的尺寸大和亚基复杂，人们对其力产生和调节的机制知之甚少。我们在此报告整个大鼠细胞质动力蛋白重链 (M(r) 532,000) 的表达和分析。全长 tDNA 由一系列部分克隆构建而成，并在 C 末端用 FLAG 表位标签或 His(6) 标签进行标记。重组多肽通过杆状病毒感染在昆虫细胞中表达，或通过瞬时转染在COS-7细胞中表达，重组蛋白大部分可溶，并表现出良好的微管结合能力。它表现出广泛的沉降曲线，表明二聚体以及更高阶多聚体的形成。在昆虫或COS-7细胞中表达的重链的测定中观察到良好的微管滑动运动活性，观察到1.2-1.8μm/秒的平均微管滑动速度，与小牛脑细胞质动力蛋白测定的速率相当。这些结果首次表明重组重链本身就能够强制产生，并且应该会导致在定义动力蛋白运动结构域方面取得快速进展。
The dyneins are a class of motor protein involved in ciliary and flagellar motility, organelle transport, and chromosome segregation, Because of their large size and subunit complexity, relatively little is known about their mechanisms of force production and regulation. We report here on the expression and analysis of the entire rat cytoplasmic dynein heavy chain (M(r) 532,000). Full-length tDNAs were constructed from a series of partial clones and tagged at the C terminus with either a FLAG-epitope tag or a His(6)-tag. The recombinant polypeptides were expressed either in insect cells by baculovirus infection or in COS-7 cells by transient transfection, The recombinant protein was mostly soluble and showed good microtubule binding. It exhibited a broad sedimentation profile, indicative of the formation of dimers as well as higher order multimers. Good microtubule gliding motility activity was observed in assays of heavy chain expressed in either insect or COS-7 cells, Average microtubule gliding velocities of 1.2-1.8 mu m/sec were observed, comparable with the rates determined for calf brain cytoplasmic dynein. These results represent the first indication that recombinant heavy chain alone is capable of force production, and should lead to rapid progress In defining the dynein motor domain.