4.2.1. Protein Expression and Purification

In this work we used the following tau4RD (Q244-E372 plus initial Met) protein variants: tau4RD C291A, C322A (here named tau4RD ΔC) and tau4RD C291A, C322A, K353C, here named tau4RD(353). The two proteins were expressed without a ffinity tag and purified as described in [30].

The ubiquitin mutants K48R, K63R, D77 were produced with the same protocol used for wild-type ubiquitin that is described in [44].

The recombinant enzymes human His-tagged E1, GST-tagged E2–25K, yeas<sup>t</sup> His-tagged Mms2, yeas<sup>t</sup> GST-tagged Ubc13 were produced as previously described [45]. In the present work, the GST tag was removed from the E2–25K protein by incubating the clean fusion protein attached to the GSH-resin with thrombin.

To obtain a ubiquitin bearing an aminoethanethiol C-terminal group (Ub-SH) required for the disulfide-coupling reaction, we first produced a chimeric protein where ubiquitin was cloned to the N-terminal of the GyrA intein in a pET22 vector. In this way, the chimeric protein has a C-terminal His-tag. The Ub-intein-His protein was produced in BL21(DE3) cells at 37 ◦C overnight in auto-inducing medium and purified by immobilized nickel a ffinity chromatography (IMAC) according to standard protocols. Cleavage of Ub-SH was obtained by incubating the clean fusion protein in a bu ffer at pH 7.5 containing: Tris-HCl 20 mM, EDTA 1 mM, cysteamine 40 mM, and Tris(2-carboxyethyl)phosphine (TCEP) 3 mM for 48 h at 10 ◦C. Ub-SH was further purified by reverse -IMAC and a superdex-75 gel filtration column when required.
