组氨酸标签野生型酵母肌动蛋白：其特性及在获得酵母肌动蛋白突变体方法中的应用。
Histidine-tagged wild-type yeast actin: its properties and use in an approach for obtaining yeast actin mutants.
作者信息
Buzan J, Du J, Karpova T, Frieden C
机构信息
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
出版信息
Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2823-7. doi: 10.1073/pnas.96.6.2823.
Wild-type and an N-terminal 6-histidine-tagged actin have each been expressed by using a yeast strain that contains the actin gene on a plasmid and not on the chromosome. Yeast strains have also been constructed that use two plasmids, one expressing the wild-type protein and the other the 6-histidine-tagged protein. Yeast cells can be grown with either plasmid alone or with both plasmids together and appear to be normal in that the growth rates of all the yeast strains are quite similar, as is the morphology of the yeast cells. The polymerization properties of the 6-histidine-tagged actin appear almost identical to wild-type actin expressed from the chromosome. When the wild-type and 6-histidine-tagged actin are coexpressed, they can be purified by standard techniques and then separated using nickel-nitrilotriacetate chromatography. The method can be used to prepare actin mutants including those that are nonfunctional or might not support yeast growth for other reasons.
野生型肌动蛋白和N端带有6个组氨酸标签的肌动蛋白，均通过使用一种酵母菌株来表达，该酵母菌株的肌动蛋白基因位于质粒上而非染色体上。还构建了使用两种质粒的酵母菌株，一种表达野生型蛋白，另一种表达带有6个组氨酸标签的蛋白。酵母细胞可以单独使用任一质粒生长，也可以同时使用两种质粒生长，并且看起来是正常的，因为所有酵母菌株的生长速率相当相似，酵母细胞的形态也是如此。带有6个组氨酸标签的肌动蛋白的聚合特性似乎与从染色体表达的野生型肌动蛋白几乎相同。当野生型肌动蛋白和带有6个组氨酸标签的肌动蛋白共表达时，它们可以通过标准技术纯化，然后使用镍-亚氨基三乙酸色谱法分离。该方法可用于制备肌动蛋白突变体，包括那些无功能的或可能由于其他原因不支持酵母生长的突变体。
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