Protein purification

Seed aggregates for addition to cells were generated with purified recombinant cysteine-free TauRD (Tau residues 244-371, C291A/P301L/C322A/V337M) to avoid the use of reducing agents that might interfere with Clu function (Supplementary Fig. ^1a). Mutation of the two cysteines in TauRD avoids the formation of intramolecular disulfide bonds that slows fibril formation⁴². Cysteine-free TauRD and TauRD-I260C were expressed as N-terminal His₆-ubiquitin fusion proteins in Escherichia coli BL21(DE3) cells transformed with the respective pHUE-TauRD plasmids via IPTG induction. The cell pellet from a 2 l culture was resuspended in 50 ml lysis buffer (50 mM PIPES-NaOH pH 6.5, 250 mM NaCl, 10 mM imidazole, 2 mM β-mercaptoethanol (βME)) supplemented with 1 mg ml⁻¹ lysozyme, Complete EDTA-free protease inhibitor cocktail (MERK) and benzonase, and incubated while gently shaking at 4 °C for 30 min. Cells were lysed by ultrasonication, and the lysate was cleared by centrifugation (1 h, 40,000 × g at 4 °C). The supernatant was loaded onto a Ni-NTA column equilibrated with lysis buffer. The column was washed with high salt buffer (50 mM PIPES-NaOH pH 6.5, 500 mM NaCl, 10 mM imidazole, 2 mM βME) and wash buffer (50 mM PIPES-NaOH pH 6.5, 250 mM NaCl, 50 mM imidazole, 2 mM βME), and His₆-ubiquitin-TauRD was eluted with elution buffer (50 mM PIPES-NaOH pH 6.5, 50 mM NaCl, 250 mM imidazole, 2 mM β-ME). The eluted fractions were collected and the salt concentration was reduced by diluting the sample (1:5) with PIPES buffer (50 mM PIPES-NaOH pH 6.5, 2 mM βME), followed by incubation with Usp2 ubiquitin protease (0.5 mg) overnight at 4 °C in order to cleave the His₆-ubiquitin tag. The cleavage mixture was applied to a Source30S cation exchange column and the TauRD protein was eluted with a 0–500 mM NaCl gradient in PIPES buffer. The TauRD protein was further purified by size exclusion chromatography (SEC) on Superdex-75 in phosphate-buffered saline (PBS). For TauRD-I260C, the buffer used for SEC contained 1 mM tris(2-carboxyethyl)phosphine (TCEP) in order to prevent the formation of disulfide bonds. Fractions containing pure protein were combined, aliquoted, and flash-frozen in liquid nitrogen for storage at −80 °C. A more detailed protocol can be found here, https://edmond.mpdl.mpg.de/imeji/collection/3psV1MQj0fmYu5KS.

Wild type FLTau (2N4R) was expressed in E. coli BL21(DE3) transformed with the Tau/pET29b plasmid via IPTG induction. The cell pellet from 6 l of culture was resuspended in 180 ml lysis2 buffer (50 mM MES-NaOH pH 6.8, 20 mM NaCl, 1 mM MgCl₂, 5 mM DTT), applied to a French press cell disruptor, and subsequently boiled for 20 min. The lysate was cleared by centrifugation (1 h, 40,000 × g at 4 °C), and the supernatant was loaded onto a Source30S cation exchange column equilibrated with lysis2 buffer. The protein was eluted with a 0–500 mM NaCl gradient and further purified by SEC on Sephacryl S-200 in 20 mM MES-NaOH pH 6.8, 20 mM NaCl, 10% glycerol. Fractions containing pure protein were combined, aliquoted, and flash-frozen in liquid nitrogen for storage at −80 °C.

Recombinant Clu (CluStrep) was purified as described (10.17504/protocols.io.bvvkn64w). Strep-tagged Clu was expressed and secreted by HEK293E-CluStrep cells cultured in FreeStyle 293 Expression Medium (Thermo Fisher Scientific) for 4 days. The conditioned medium was then separated from the cells by centrifugation. For chromatographic purification, the medium was first dialyzed against wash buffer (20 mM Na acetate pH 5.0). After removal of the precipitate by centrifugation, the supernatant was passed over a HiTrap SP XL cation exchange column. The column was washed with 10 column volumes (CV) denaturing buffer (20 mM Na acetate pH 5.0, 6 M urea), followed by 5 CVs wash buffer. For protein elution, a 0–500 mM NaCl gradient in wash buffer was applied. Clu-containing fractions were further purified by SEC on Superdex-200 in 20 mM Na acetate pH 5.0, 100 mM NaCl, 1 mM EDTA. Fractions containing pure Clu were concentrated, and the buffer was exchanged to PBS using a Nap5 (GE Healthcare) column. Aliquots were flash-frozen in liquid nitrogen for storage at −80 °C.

Recombinant human α-Synuclein (α-Syn, A53T) was purified essentially as described⁸⁹ (10.17504/protocols.io.btynnpve). In brief, E. coli BL21(DE3) cells were transformed with the pT7-7 α-Syn A53T plasmid. Protein expression was induced with 1 mM IPTG for 4 h at 37 °C. Bacteria were harvested and the pellet was lysed in high salt buffer (750 mM NaCl, 50 mM Tris-HCl pH 7.6, 1 mM EDTA). The lysate was sonicated for 5 min and boiled subsequently. The boiled suspension was centrifuged, the supernatant dialyzed against 50 mM NaCl, 10 mM Tris-HCl pH 7.6, and 1 mM EDTA and purified by SEC on Superdex 200 in the same buffer. Fractions were collected and those containing α-Syn were combined. The combined fractions were applied onto an anion exchange column (MonoQ). α-Syn was purified by a gradient ranging from 50 mM to 1 M NaCl. Fractions containing α-Syn were combined and dialyzed in 150 mM KCl, 50 mM Tris-HCl pH 7.6. Aliquots were stored at −80 °C.