KrishnaKumar V Guru, Gupta Sharad
a Biological Engineering , Indian Institute of Technology Gandhinagar, Palaj , Gandhinagar , India.
Prep Biochem Biotechnol. 2017 May 28;47(5):530-538. doi: 10.1080/10826068.2016.1275012. Epub 2017 Jan 3.
Tau is an intrinsically disordered protein responsible for maintaining the structure and stability of axonal microtubules. However, in certain disease conditions including Alzheimer's disease, tau protein may undergo biochemical and structural changes to form intracellular aggregates. Since tau is a proline- and arginine-rich eukaryotic protein, heterologous expression in Escherichia coli often results in poor yield and has been a major technical challenge. In the current work, we have improved the expressed yield of tau by overcoming codon bias problem and established a simplified protocol for efficient extraction. The reported method has two distinct features: (i) enhanced tau expression (upto eightfold) by supplementing deficient tRNAs that aid in rapid translation and (ii) direct boiling of expressed E. coli cells to extract tau with no separate cell lysis step. We further demonstrate that tau extracted by the direct boiling method is similar to tau purified by size-exclusion chromatography exhibiting similar structural and biophysical characteristics including aggregation propensity. Since morphologies and in vitro toxicity of fibrillar tau aggregates were also similar, tau extracted by the one-step direct boiling method can be used for tau aggregation assays without any additional purification.
tau蛋白是一种内在无序的蛋白质，负责维持轴突微管的结构和稳定性。然而，在包括阿尔茨海默病在内的某些疾病状态下，tau蛋白可能会发生生化和结构变化，形成细胞内聚集体。由于tau是一种富含脯氨酸和精氨酸的真核蛋白，在大肠杆菌中进行异源表达往往产量较低，这一直是一个主要的技术挑战。在当前的工作中，我们通过克服密码子偏好问题提高了tau的表达产量，并建立了一种简化的高效提取方案。所报道的方法有两个显著特点：（i）通过补充有助于快速翻译的缺陷型tRNA来提高tau的表达（高达八倍）；（ii）直接煮沸表达tau的大肠杆菌细胞来提取tau，无需单独的细胞裂解步骤。我们进一步证明，通过直接煮沸法提取的tau与通过尺寸排阻色谱法纯化的tau相似，表现出相似的结构和生物物理特性，包括聚集倾向。由于纤维状tau聚集体的形态和体外毒性也相似，通过一步直接煮沸法提取的tau可用于tau聚集测定，无需任何额外的纯化。
