Tau expression and purification

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

Buffer B

Buffer C