4.3. Expression and Purification of Recombinant Proteins (E. coli and SF9 Tau)

All plasmids were verified by Sanger sequencing prior to protein production. Human Tau isoforms and variants (2N4R, 1N4R, 0N4R, TauRD, N-term) were expressed in E. coli BL21 Star (DE3) (Invitrogen, Waltham, MA, USA) as previously described [37]. Protein expression was induced with 0.5 mM IPTG at OD600 = 0.6 for ~3 h at 37 °C. Cells were harvested, resuspended in lysis buffer (20 mM MES, 1 mM EGTA, 0.2 mM MgCl₂, 1 mM PMSF, 5 mM DTT, protease inhibitors (Pierce Protease Inhibitor Mini Tablets, EDTA-free), and lysed using a French press. After initial purification by adding 500 mM NaCl and boiling at 95 °C for 20 min, cell debris was removed by centrifugation and the supernatant was dialyzed against a low salt buffer (Buffer A: 20 mM MES, 50 mM NaCl, 1 mM MgCl₂, 1 mM EGTA, 2 mM DTT, 0.1 mM PMSF, pH 6.8), filtered (0.22 µm membrane filter), run through a cation exchange column (HiTrap SP HP, 5 mL, GE Healthcare, Chicago, IL, USA), and eluted with a high salt buffer (Buffer B: 20 mM MES, 1000 mM NaCL, 1 mM MgCl₂, 1 mM EGTA, 2 mM DTT, 0.1 mM PMSF, pH 6.8). Fractions containing Tau were pooled, concentrated using spin column concentrators (Pierce Protein concentrators; 10–30 kDa MWCO, Thermo Fischer Scientific, Waltham, MA, USA), and run through a size exclusion column (Superose 6 10/300, GE Healthcare, Chicago, IL, USA). Fractions containing purified monomeric Tau were concentrated as before and buffer exchanged to PBS, 1 mM DTT, pH 7.4. The final Tau concentration was measured with BCA assay (BCA kit, PIERCE), and the protein was stored at −80 °C.

Phosphorylated Tau (P-Tau and P-TauΔK280) was expressed in insect (SF9) cells by infection with a recombinant baculovirus containing a pTriEx-Tau plasmid that encodes human full-length Tau (2N4R or TauΔK280) with a N-terminal Strep-Tag [51]. After cell lysis, the cleared lysate was applied to a 10 mL Strep-Tactin column (Iba) and a gel filtration column (Superdex200 16,600, GE Healthcare, Chicago, IL, USA). The purified protein was stored in PBS containing 1 mM TCEP at −80 °C.

Nup98-FG and Nup98-FG/FS plasmid constructs were bought from GenScript (Piscataway, NJ, USA) and expressed and purified as described before [52]. Briefly, Nup98-FG represents the N-terminal FG-domain of Nup98 (aa 1-498), with one additional cysteine at the N-terminus to facilitate covalent binding to the gold surface of the SPR chip; Nup98-FG/FS is the full F/S mutant of Nup98-FG.