Hiload 16 60 desalting column

pET15b-Tau recombinant T7lac expression plasmid was transformed into competent E. coli BL21 (DE3) bacterial cells. A small-scale culture was grown in LB medium at 37°C and was added at 1:10 v/v to 1 L of a modified M9 medium containing MEM vitamin mix 1X (Sigma-Aldrich), 4 g of glucose, 1 g of ¹⁵N-NH₄Cl (Sigma-Aldrich), 0.5 g of ¹⁵N-enriched Isogro (Sigma-Aldrich), 0.1 mM CaCl₂, and 2 mM MgSO₄. Recombinant ¹⁵N Tau production was induced with 0.5 mM IPTG when the culture reached an optical density at 600 nm of 0.8. Proteins were first purified by heating the bacterial extract, obtained in 50 mM NaPi, pH 6.5, 2.5 mM EDTA, and supplemented with complete protease inhibitor cocktail (Sigma-Aldrich), 15 min at 75°C. The resulting supernatant was next passed on a cation exchange chromatography column (Hitrap SP sepharose FF, 5 mL, Cytiva) with 50 mM NaPi, pH 6.5, and eluted with a NaCl gradient. Tau proteins were buffer-exchanged against 50 mM ammonium bicarbonate (Hiload 16/60 desalting column, Cytiva) for lyophilization. The same protocol⁶⁹ was used to produce and purify Tau 2N3R isoform, Tau[244–368] (designated MTBD, also called K18 fragment), chimeric Tau[244–368] with two PHF6 or PHF6∗ peptide sequences instead of PHF6∗ and PHF6 sequences (Figure S10A) and Tau [208–324].