Heterologous Expression,purification And Aggregation Characteristics Of Alzheimer’s Disease Pathogenic Proteins Aβ42 And Tau

The typical pathological feature of Alzheimer’s disease(AD)is that β-amyloid(Aβ)protein aggregates to form extracellular amyloid plaques and hyperphosphorylated Tau protein aggregates to form intracellular neurofibrillary tangles(NFTs).Aβ is a peptide produced by the hydrolysis of amyloid precursor protein by β-hydrolase and y-hydrolase.When the specificity of β-hydrolase and y-hydrolase is reduced,a series of N-terminal or C-terminal extended or truncated Aβvariants will be produced.Related studies have shown that the aggregation characteristics of these Aβ variants are different,but for heterologous residues at the N-terminus.The effect of aggregation characteristics has not been systematically studied.Our previous studies showed that the C-terminally coupled His6 tag had little effect on the aggregation characteristics of Aβ42.However,the effect of N-terminally coupled His6 tag on the aggregation properties and toxicity of Aβ42 protein is unknown.Based on the above reasonss,a recombinant Aβ42 protein(His6-Aβ42)with a His6 tag modification at the N-terminus was purified by E.coli expression.Then,the aggregation characteristics of His6-Aβ42 were studied by thioflavin T fluorescence experiment and atomic force microscope.It was found that the addition of His6 at the N-terminal reduced the aggregation characteristics of Aβ42,and that His6-Aβ42 formed more amorphous aggregates,which was significantly different from the normal fibril formed by Aβ42.However,the cytotoxicity experiment proved that the above-mentioned amorphous aggregates showed stronger toxicity to PC 12 cells than Aβ42.The above results indicate that the presence of the N-terminal heterologous residues,that is,the presence of the His6 tag,has a significant effect on the aggregation characteristics and toxicity of the Aβ42 protein.The disease caused by the abnormal aggregation and precipitation of Tau protein is called tauopathies,of which AD is the most common type.The microtubule binding domain(MBD)of Tau can bind to tubulin to maintain the stability of microtubules,which is the core region of the Tau protein.Studies have confirmed that the Tau protein 306-378 peptide(Tau306-378)is the core region that drives its aggregation,that is,R3 and R4 specific sequences and 10 amino acid residues extending backward from the C-terminus of the R4 sequence.In this study,Tau&MBD and Tau306-378 were first obtained by expressing pET22b vector in E.coli,and then purified by Ni+affinity chromatography.The experimental studies using thioflavin T fluorescence staining proved that the two recombinant proteins have good aggregation characteristics.Atomic force microscopy experiments showed that Tau&MBD aggregated to form a typical amyloid fibril,and cytotoxicity experiments showed that it has strong toxicity.The above shows,the core fragments of these two Tau proteins obtained in this study have good biological activity and can be used for in vivo and in vitro physiological,biochemical,and toxicity studies,as well as for the screening of various types of aggregation inhibitors.