Recombinant tau expression and purification
1. Transformation
· Transform 50 ul Escherichia coli BL21 (DE3) with pRK172-2N4R or -2N3R plasmid
· Incubate on ice for 30min
· Heatshock @ 42°C for 90 sec
· Recovery in 400 ul 2xTY for 1 h @ 37°C
· Plate 100 ul onto TYE+ampicillin plate
· Incubate at 37°C overnight
2. Cell growth
· Add ampicillin to 2L flask 2xTY containing 500ml of medium plus 5mM MgCl2 to final conc. 100 ug/ml
· Pipette 10 ml sterile 2xTY onto plate of bacteria, resuspend cells thoroughly
· Inoculate 500ml medium with 5 ml of resuspended bacteria. Therefore, one plate will inoculate two 2L flasks.
· Grow @ 37°C to OD600 ~0.8 (~2.5 h)
· Induce with 0.4 mM IPTG
· Incubate @ 37°C for 3hr
· Harvest cells by centrifugation, 5k RPM for 20 min
· Store @ -20°C
3. Lysis
· Resuspend cell pellet in 15 mL buffer A / L culture
· Lyse using probe sonicator on ice (3 min of working time; 5 s on/ 6 s off; 40% amplitude)
· Add DNAse (Sigma) to 40 ug/mL and RNAse (sigma) to 10 ug/mL, incubate 5 min
· Spin 18k RPM, 30 min at 4°C
· Pass supernatant through 0.45 mM syringe filter
3. Cation exchange (Hitrap CaptoS column (GE Healthcare))
· Wash HiTrap CaptoS column with 5 CV MPW, then 5 CV buffer A, then 5 CV buffer B, then 5 CV buffer A
· Load cleared lysate onto column at 5 mL/min
· Run HiTrap CaptoS column program (5 mL/min; 5 mL fractions; 10 CV buffer A washing, followed by 50-500 mM NaCl gradient over 10 CV (Caution: The running program here is for reference. Best running program needs to be optimized accordingly)
· Wash HiTrap CaptoS column with 5 CV buffer B, then 5 CV buffer A, then 5 CV MPW, then 5 CV 20% EtOH
4. Ammonium sulphate precipitation
· Pool best fractions according to the results of Tris-Glycine SDS-PAGE (4–20%), and precipitate with 38% ammonium sulphate for 30 min on ice
· Spin 15k RPM, 20 min
· Store pellet at -20°C
5. Gel filtration (HiLoad 16/600 Superdex 200 pg (GE Healthcare))
· Equilibrate column with 2 CV buffer C
· Resuspend pellet in 2 mL buffer C, centrifuge at 100,000 × g at 4°C for 1h.
· Load the supernatant onto gel filtration column at 1.0 mL/min
· Run gel filtration program (1.0 mL/min; collecting 0.8 mL/tube fractions after 0.29 CV)
6. Concentration
· After SDS-PAGE, the fractions of pure tau protein with least degradation were collected and spin concentrated to 3.0 mg/mL (Vivaspin® 20, 10 kDa MWCO Polyethersulfone)
· Freshly purified proteins were used for Heparin-induced filament assembly. The left proteins were aliquoted and snap-frozen for storage at −20 °C.
Buffer A
50 mM MES pH 6.5 |
50 mM NaCl |
10 mM EDTA |
5 mM MgCl2 |
5 mM TCEP |
0.1 mM AEBSF |
0.03 mM Chymostatin |
cOmplete EDTA-free Protease Inhibitor Cocktail (Roche) |
Buffer B
50 mM MES pH 6.5 |
1 M NaCl |
10 mM EDTA |
5 mM MgCl2 |
5 mM TCEP |
0.1 mM AEBSF |
0.03 mM Chymostatin |
cOmplete EDTA-free Protease Inhibitor Cocktail (Roche) |
Buffer C
1x PBS |
5 mM TCEP |
0.1 mM AEBSF |
0.015 mM Chymostatin |
cOmplete EDTA-free Protease Inhibitor Cocktail (Roche) |