Expression and purification of tau protein and its frontotemporal dementia variants using a cleavable histidine tag

Abstract

Recombinant tau protein is widely used to study the biochemical, cellular and pathological aspects of tauopathies, including Alzheimer's disease and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTPD-17). Pure tau in high yield is a requirement for in vitro evaluation of the protein's physiological and toxic functions. However, the preparation of recombinant tau is complicated by the protein's propensity to aggregate and form truncation products, necessitating the use of multiple, time-consuming purification methods. In this study, we investigated parameters that influence the expression of wild type and FTPD-17 pathogenic tau, in an attempt to identify ways to maximise expression yield. Here, we report on the influence of the choice of host strain, induction temperature, duration of induction, and media supplementation with glucose on tau expression in Escherichia coli. We also describe a straightforward process to purify the expressed tau proteins using immobilised metal affinity chromatography, with favourable yields over previous reports. An advantage of the described method is that it enables high yield production of functional oligomeric and monomeric tau, both of which can be used to study the biochemical, physiological and toxic properties of the protein.

The C291R Tau Variant Forms Different Types of Protofibrils

“…As cysteine-291 is important for tau aggregation (Bhattacharya et al, 2001;Crowe et al, 2013;Soeda et al, 2015), we asked if the C291R mutation affects aggregation propensity. Each tau-K18 construct was mixed with a 50% mass concentration of heparin and incubated at 37 • C without shaking for 48 h. Negative-stain TEM analysis showed that tau-K18 WT aggregated into fibrils ( Figure 3A) in agreement with previous reports (Barghorn et al, 2000;Barghorn and Mandelkow, 2002;Karikari et al, 2017). These fibrils were 30-45 nm wide and 150-300 nm long.…”

“…We generated the pProEx-HTa-Myc-6 × His-K18 plasmid carrying the C291R mutation (TGT > CGT, the same codon change previously reported by Marshall et al, 2015) by site-directed mutagenesis using a WT tau-K18 plasmid as a template (Karikari et al, 2017(Karikari et al, , 2019a. Protein expression and purification were achieved using our previously-characterized recombinant tau production protocols (Karikari et al, 2017(Karikari et al, , 2019aHill et al, 2019). The purified His-tagged tau-K18 constructs were used directly in functional experiments since the His-tag does not appear to affect aggregation (Huseby et al, 2019).…”

“…The microtubule-binding region of tau (amino acids 244-372 of full-length tau-441; also known as tau-K18) was cloned into a pProEx-HTa plasmid and the C291R mutation introduced by site-directed mutagenesis and sequence-verified. This genetic construct was transformed into BL21(DE3)*pRosetta Escherichia coli, expressed and purified following a previously-described protocol also used for preparing wild type tau-K18 (Karikari et al, 2017). FIGURE 2 | Circular dichroism (CD) profiles of tau-K18 WT (A) and C291R (B) monomers.…”

“…Tau-K18 is generally unfolded but adopts β-sheet conformation with increasing aggregation (Kumar et al, 2014;Karikari et al, 2017). Specific MAPT mutations have distinct effects on this process by either increasing or decreasing the propensity to form β-sheet structures (Barghorn et al, 2000;Combs and Gamblin, 2012;Karikari et al, 2019a).…”

A novel small molecule screening platform for disrupting toxic tau oligomers in cells

“…The formation of these toxic tau oligomers has been associated with mutations and overexpression of numerous chaperone proteins [36][37] , highlighting the importance of other components of the tau-protein interactome in the pathogenesis of tauopathies. Capturing this complexity in an in vitro setting is extremely difficult, if even possible, and established protocols for aggregating purified tau protein into oligomers, PHFs, and NFTs have, not surprisingly, been shown to produce different tau assemblies depending on aggregation conditions 17,31,34,[38][39][40][41] . Critically, no specific toxic tau species has been isolated or identified to date 21,[42][43] .…”