Recombinant 15N-labeled Tau Protein Production

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 1× (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 CaCl_2, and 2 mM MgSO₄. Recombinant ¹⁵N tau (NCBI reference number NP_005901.2) 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 inhibitors 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. Detailed procedure can be found in (62 ).
K18 expression and purification were performed according to (63 (link)).