Background

Tau is traditionally defined as a microtubule binding protein; however, in human diseases Tau can dissociate from axonal microtubules and mislocalize to other neuronal compartments including the soma, dendrites, and synapses, where interactions with non-microtubule proteins and structures drive neuronal dysfunction (Iqbal et al., 2016; Wang and Mandelkow, 2016; Zhou et al., 2017; McInnes et al., 2018). Although Tau aggregates in the form of neurofibrillary tangles are commonly found in post-mortem diseased brain tissue, studies suggest that soluble Tau, not aggregated Tau, is largely responsible for neuronal dysfunction (Crimins et al., 2012; Polydoro et al., 2014; Koss et al., 2016). As such, investigating the soluble functions of Tau in disease, such as identifying protein-protein interaction partners, is therefore of critical importance to target Tau dysfunction.

Purifying soluble Tau protein has been challenging since many ectopically expressed recombinant proteins aggregate into insoluble inclusion bodies in bacteria. Tau furthermore contains aggregation-prone motifs, making purifying recombinant Tau even more difficult. We have therefore developed a protocol that overcomes these obstacles by optimizing two key aspects of this process: first, we express Tau as a fusion protein with an N-terminal Glutathione S-transferase (GST) tag to enhance solubility of the protein, and utilize a mild induction paradigm to minimize the formation of insoluble Tau aggregates. We chose GST over other fusion proteins because it is relatively small, robustly enhances protein solubility and stability, can be easily cleaved, and is relatively inexpensive to purify. Second, our purification protocol utilizes dual affinity tag purification of both an N-terminal GST tag and a C-terminal 8xHis tag (Figure 1); because this purification protocol requires both N- and C-terminal tags to be available to bind affinity resin, the protocol therefore specifically enriches for soluble, non-aggregated protein (in the event of aggregation, at least one of these epitopes is hidden within the aggregate). Following the first purification step against the N-terminal GST tag, the GST fusion protein is cleaved using PreScission protease, resulting in a final product of purified Tau protein that contains only an additional eight histidine residues (8xHis tag) at its C-terminal domain that can also be used for downstream applications including pull-down experiments or detection using anti-His antibodies. This method is versatile and can be easily modified to purify different isoforms of Tau, Tau carrying point mutations, or Tau truncations by altering the pGEX_GST-Tau0N4R-8xHis plasmid using site-directed mutagenesis or traditional cloning methods. We have successfully utilized this protocol to produce various isoforms of Tau, including with point mutations or domain truncations, for use in in vitro binding assays with synaptic vesicles (Zhou et al., 2017) and as bait in protein-protein interaction studies (McInnes et al., 2018).


Figure 1. Schematic overview of the recombinant GST-Tau-8xHis fusion protein and purification steps