中国生物工程杂志

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.