Tau蛋白核心片段306~378的异源表达、纯化及聚集特性验证*

Tau是一种微管相关蛋白,其生理功能是与微管蛋白结合促进其聚合形成微管并维持微管的稳定。由于Tau蛋白异常聚集沉淀而导致的疾病被称为Tau蛋白病,其中阿尔茨海默病是最常见的一种类型。全序列Tau含有441个氨基酸残基,其中306~378肽段(Tau_306-378)为驱动其聚集的核心区域。Tau_306-378包含R3和R4微管结合序列以及从R4序列C端向后延伸的10个氨基酸残基。首先利用pET22b载体在大肠杆菌中表达获得了Tau_306-378,然后用镍亲和层析进行纯化,终产量约为10.35mg/L。利用SDS-PAGE、Western blot和基质辅助激光解析电离飞行时间质谱依次对Tau_306-378进行了鉴定。其中SDS-PAGE和基质辅助激光解析电离飞行时间质谱的研究结果表明,表达的Tau_306-378主要以单体形式存在,但同时含有部分二聚体。最后,硫黄素T荧光染色实验显示该重组蛋白具有良好的聚集特性,可用于体外Tau蛋白的聚集特性、毒性及相关抑制剂开发的研究。

Heterologous Expression,Purification and Aggregation Characterization of Tau Core Fragment 306-378

Tau is one of the microtubule-associated proteins. Its physiological function is to promote microtubule assembly and maintain microtubule stability. The phosphorylated Tau easily aggregated and subsequently caused a series of tauopathies. Of them, Alzheimer's disease is a common type of tauopathies. Six subtypes of Tau are also found in human brain because of different mRNA splicing of the same gene. They are respectively composed of 352, 381, 383, 410, 412 and 441 residues. The molecular weight of the six subtypes is among 48-67kDa, the R1, R2, R3 and R4 are the microtubule binding domains, which can bind to microtubule and maintain microtubule stability. Compared with the full-length Tau, the truncated Tau isoforms, such as R1-R4, are more likely to aggregate and have stronger aggregation kinetics. For example, the microtubule binding domain of R3 and R4 and the following 10 amino acid at the C-terminal of R4 sequence are the core peptide of the paired helical fibrils (PHFs), which has been proven to drive Tau aggregation. And its fibrillary 3D structure was also identified using micro-electron diffraction of Cryo-electron microscope. Herein, the core fragment 306-378 (Tau_306-378) of Tau was heterogeneously expressed in Escherichia coli, and the purified Tau_306-378 was obtained by Ni⁺ chelated affinity chromatography. The final production yield was about 10.35mg/L. Then, the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot proved that Tau_306-378 was expressed successfully. Moreover, the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was further used to confirm the molecular weight of Tau_306-378. It was proved that most of the Tau_306-378 were in the form of monomers. In addition, a small portion of dimers were also found in the protein sample, which is caused by the formation of disulfide bond among two monomers. In order to explorethe effect of induction temperature on the production of the target protein, four different induction temperatures (16℃, 25℃, 30℃ and 37℃) were investigated. The results showed that the expression of Tau_306-378 was the highest at 30℃, followed by 25℃ and 16℃. However, the expression of Tau_306-378 was the lowest at 37℃. The optimal inducing condition of Tau_306-378 were identified: inducing at 30℃ for 16-20h with 0.5mmol/L of isopropyl-β-D-thiogalactopyranoside. Finally, thioflavin T fluorescence staining experiments were performed to probe its fibrillogenesis. The results of thioflavin T fluorescence experiments showed that the trend of aggregation dynamics presents a typical S-shaped curve. That is, the recombinant Tau_306-378 has good aggregation characteristics, which could be used to study the aggregation characteristics and toxicity of Tau in vitro, as well as in the screening of various inhibitors against Tau fibrillogenesis.