Purification of modified mammalian actin isoforms for in vitro reconstitution assays.
Purification of modified mammalian actin isoforms for in vitro reconstitution assays.
复制标题
纯化修饰的哺乳动物肌动蛋白亚型用于体外重构测定。
DOI：
10.1016/j.ejcb.2023.151363
复制
发表时间：
2023
影响因子：
6.6
通讯作者：
Jansen,Silvia
中科院分区：
文献类型：
--
作者：
Kast,DavidJ;Jansen,Silvia
使用纯化的肌动蛋白在体外重建试验大大提高了我们的理解细胞骨架动力学和肌动蛋白结合蛋白的调节。然而，早期的纯化方法包括获得纯肌动蛋白的苛刻条件，并且通常不包括分离的肌动蛋白单体的正确成熟和专性修饰。新的见解的折叠要求和N-末端加工的肌动蛋白，以及更好地了解肌动蛋白与单体螯合蛋白，如DNaseI，profilin和凝溶胶蛋白的相互作用，导致开发更温和的方法来获得纯的重组肌动蛋白亚型与已知的专性修饰。本文综述了从组织和细胞中分离天然折叠的内源性和重组肌动蛋白的方法。我们进一步强调了每种方法的使用和局限性，并描述了如何实施这些方法来研究肌动蛋白PTM，疾病相关的肌动蛋白突变和新型肌动蛋白样蛋白。
In vitro reconstitution assays using purified actin have greatly improved our understanding of cytoskeletal dynamics and their regulation by actin-binding proteins. However, early purification methods consisted of harsh conditions to obtain pure actin and often did not include correct maturation and obligate modification of the isolated actin monomers. Novel insights into the folding requirements and N-terminal processing of actin as well as a better understanding of the interaction of actin with monomer sequestering proteins such as DNaseI, profilin and gelsolin, led to the development of more gentle approaches to obtain pure recombinant actin isoforms with known obligate modifications. This review summarizes the approaches that can be employed to isolate natively folded endogenous and recombinant actin from tissues and cells. We further emphasize the use and limitations of each method and describe how these methods can be implemented to study actin PTMs, disease-related actin mutations and novel actin-like proteins.