Rescue of a Chlamydomonas inner-arm-dynein-deficient mutant by electroporation-mediated delivery of recombinant p28 light chain
Rescue of a Chlamydomonas inner-arm-dynein-deficient mutant by electroporation-mediated delivery of recombinant p28 light chain
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DOI：
10.1002/cm.10075
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发表时间：
2002-12-01
影响因子：
--
通讯作者：
Kamiya, R
中科院分区：
文献类型：
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作者：
Hayashi, M;Yanagisawa, H;Kamiya, R
我们最近已经证明，可以通过电穿孔将兔肌动蛋白导入缺乏常规肌动蛋白的衣藻ida5突变株中，并挽救其突变表型[Hayashi等，2001：cell Motil]。细胞骨架49：146-153]。在这项研究中，我们探索了利用电穿孔技术进行重组蛋白功能检测的可能性。在大肠杆菌中表达了内臂动力蛋白的p28轻链，纯化至均一，并通过电穿孔导入缺乏该轻链的非运动性突变体ida4oda6中。由于该蛋白不溶于先前研究中使用的低离子强度溶液，因此在有钙离子存在的情况下，在生理离子强度下进行电穿孔。大多数细胞在电穿孔后脱落鞭毛。重组p28在3h内发生重排，高达30%的细胞开始活动，表明导入的p28仍具有功能活性。荧光标记的p28同样有效；在这种情况下，沿着鞭毛观察到荧光。钙离子和偏转的存在似乎对有效的蛋白质传递很重要，因为带有鞭毛脱落机制缺陷的FA1突变的三个突变体恢复的运动性非常差。重组蛋白和突变体的其他组合也得到了类似的结果。因此，本研究证明了电穿孔技术用于重组蛋白活性检测的可行性。(C)2002年Wiley-Liss，Inc.
We have recently shown that rabbit actin can be introduced by electroporation into the Chlamydomonas ida5 mutant lacking conventional actin and rescue its mutant phenotype [Hayashi et al., 2001: Cell Motil. Cytoskeleton 49:146-153]. In this study, we explored the possibility of using electroporation for functional assay of a recombinant protein. The p28 light chain of inner-arm dyneins was expressed in Escherichia coli, purified to homogeneity, and introduced by electroporation into a non-motile mutant ida4oda6 that lacks it. Because this protein was insoluble in the low ionic strength solution used in the previous study, electroporation was 21 performed at physiological ionic strength in the presence of Ca2+. Most cells shed their flagella after electroporation. Reflagellation took place within 3 h and up to 30% of the cells became motile, indicating that the introduced p28 retained its functional activity. Fluorescently-labeled p28 was equally effective; in this case fluorescence was observed along the flagella. The presence of Ca2+ and deflagellation appeared to be important for efficient protein delivery, because a triple mutant with the fa1 mutation deficient in the flagellar shedding mechanism recovered motility only very poorly. Similar results were obtained with other combinations of recombinant proteins and mutants. This study thus demonstrates the feasibility of using electroporation for activity assays of recombinant proteins. (C) 2002 Wiley-Liss, Inc.