Production of recombinant tau oligomers in vitro

Abstract

The pathological aggregation of the tau protein is a common characteristic of many neurodegenerative diseases. There is strong interest in characterizing the potentially toxic nature of tau oligomers. These nonfibrillar, soluble multimers appear to be more toxic than neurofibrillary tangles made up of filamentous tau. However, reliable production, purification, and verification of tau oligomers can provide certain challenges. Here, we provide a series of methods that address these issues. First, recombinant tau is produced using Escherichia coli, purified through affinity, size-exclusion, and anion-exchange chromatography steps and quantified using an SDS Lowry protein quantitation assay. Aggregation of tau is induced using arachidonic acid, and oligomers are purified by centrifugation over a sucrose step gradient. Finally, we describe a sandwich enzyme-linked immunosorbent assay that utilizes the tau oligomerspecific TOC1 antibody to confirm the presence of oligomeric tau. Together, these steps provide a very simple and reliable method for producing tau oligomers that can be used in downstream applications.

Defined Tau Phosphospecies Differentially Inhibit Fast Axonal Transport Through Activation of Two Independent Signaling Pathways

“…Recombinant tau proteins in Figure 1 were prepared as previously described (Tiernan et al, 2016 ; Combs et al, 2017 ). Single or multiple pseudophosphorylation point mutations were introduced into a cDNA construct encoding full-length hTau40 using Quikchange II XL Site-Directed Mutagenesis Kit (Agilent) and confirmed by Sanger sequencing.…”

EFhd2 Affects Tau Liquid–Liquid Phase Separation

“…The final volume was 250μ L and concentration was kept below 3μ g/μL to prevent EFhd2 spontaneous self-aggregation. GFP tagged (Tau-GFP) and untagged (Tau) recombinant tau (Accession # P10636-8) proteins with C-terminal 6x poly-histidine tags were produced and purified as previously described (Combs et al, 2017). Briefly, T7 express E. coli cells were induced to express proteins with IPTG, followed by collection of cells and lysis of the cell pellet.…”

Tau is not necessary for amyloid-β–induced synaptic and memory impairments

“…Samples were assayed in total tau and oligomeric tau sELISAs using methods similar to those previously described (80)(81)(82). Recombinant tau proteins were generated as described previously (83). Monomeric tau-and arachidonic acid-induced aggregates were produced as described (83).…”

“…Recombinant tau proteins were generated as described previously (83). Monomeric tau-and arachidonic acid-induced aggregates were produced as described (83). For total tau assays, the capture antibody was Tau12 (aa8-21, catalog AB_2721192) (80,84) and 100 μg lysate protein was used.…”

“…The polyclonal pan-tau antibody, R1 (catalog AB_2832929) (87), was used for detection of captured tau. Monomeric tau-and arachidonic acid-induced aggregates were produced as described (83). Recombinant protein standards consisting of monomeric tau (10-1.3 nM) or aggregated tau (0.16-1.3 nM) were used to estimate the level of tau present in total tau assays or oTau assays,…”