Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.
The Children's Hospital of Philadelphia Research Institute, Philadelphia, PA 19104.
Proc Natl Acad Sci U S A. 2022 Oct 11;119(41):e2209150119. doi: 10.1073/pnas.2209150119. Epub 2022 Oct 5.
Actin is the most abundant protein in the cytoplasm of eukaryotic cells and interacts with hundreds of proteins to perform essential functions, including cell motility and cytokinesis. Numerous diseases are caused by mutations in actin, but studying the biochemistry of actin mutants is difficult without a reliable method to obtain recombinant actin. Moreover, biochemical studies have typically used tissue-purified α-actin, whereas humans express six isoforms that are nearly identical but perform specialized functions and are difficult to obtain in isolation from natural sources. Here, we describe a solution to the problem of actin expression and purification. We obtain high yields of actin isoforms in human Expi293F cells. Experiments along the multistep purification protocol demonstrate the removal of endogenous actin and the functional integrity of recombinant actin isoforms. Proteomics analysis of endogenous vs. recombinant actin isoforms confirms the presence of native posttranslational modifications, including N-terminal acetylation achieved after affinity-tag removal using the actin-specific enzyme Naa80. The method described facilitates studies of actin under fully native conditions to determine differences among isoforms and the effects of disease-causing mutations that occur in all six isoforms.
肌动蛋白是真核细胞质中含量最丰富的蛋白质，它与数百种蛋白质相互作用，执行基本功能，包括细胞运动和胞质分裂。许多疾病是由肌动蛋白突变引起的，但如果没有一种可靠的方法来获得重组肌动蛋白，研究肌动蛋白突变体的生物化学性质是很困难的。此外，生化研究通常使用组织纯化的α-肌动蛋白，而人类表达的六种同工型几乎完全相同，但具有专门的功能，并且难以从天然来源中单独获得。在这里，我们描述了一种解决肌动蛋白表达和纯化问题的方法。我们在人 Expi293F 细胞中获得了高产量的肌动蛋白同工型。沿着多步纯化方案进行的实验证明了内源性肌动蛋白的去除和重组肌动蛋白同工型的功能完整性。内源性和重组肌动蛋白同工型的蛋白质组学分析证实了存在天然的翻译后修饰，包括使用肌动蛋白特异性酶 Naa80 去除亲和标签后获得的 N 端乙酰化。所描述的方法促进了在完全天然条件下对肌动蛋白的研究，以确定同工型之间的差异以及发生在所有六种同工型中的致病突变的影响。
