Purification of recombinant Tau Repeat Domain (TauRD) from Escherichia coli

Thaw RbCl-competent Escherichia coli Bl21 cells (DE3) On ice.

Add 1µL of pHUE-TauRD plasmid (His 6 -ubiquitin-TauRD) and incubate 0h 30m 0s On ice.

Heat shock 0h 0m 45s at 42°C.

Incubate On ice 0h 2m 0s, then add 850µL Lysogeny broth (LB) or Super Optimal broth with Catabolite repression (SOC) medium.

Shake for 1h 0m 0s at 37°C.

Centrifuge for 0h 5m 0s at 3000x g,0h 0m 0s and remove most of the supernatant.

Resuspend the pellet with the remaining supernatant and plate the bacteria on LB/Ampicillin agar plates and incubate 1h 0m 0s at 37°C.

Prepare preculture: Scrap all colonies with the scraper and inoculate 25mL-50mL LB/Ampicillin. Shake at 37°C for 4-6h 0m 0s.

Measure OD₆₀₀ of the preculture and inoculate two flasks with 1L of TB media each to an

OD₆₀₀ = 0.05.

Shake flasks at 37°C until approx. OD₆₀₀  = 0.5-0.8. (2 - 4h 0m 0s)

Add isopropyl β-D-1-thiogalactopyranoside (IPTG) at final concentration of 0.4millimolar (mM).

Shake flasks 4h 0m 0s at 37°C.

Centrifuge bacterial culture at 4000rpm,0h 0m 0s for 1h 0m 0s. Discard supernatant. Cell pellets can be stored at -80°C.

Resuspend the cell pellets with lysis buffer (50mL lysis buffer/2L bacteria culture) supplemented with Complete EDTA-free protease inhibitor cocktail (Merck) and benzonase.

Add 1 lysozyme and incubate gently shaking for 0h 30m 0s at 4°C.

Sonicate lysate On ice, 5 cycles 0h 0m 30s ON, 0h 1m 30s OFF.

Centrifuge lysate at 40000x g,0h 0m 0s 1h 0m 0s at 4°C.

Prepare Ni-NTA column by transferring 10mL Ni-NTA resin slurry to a column (5mL column bed). Wash Ni-NTA column with 10 column volumes (CV, 50mL) water and equilibrate with 10 CV (50mL) lysis buffer.

Load lysate supernatant to Ni-NTA column.

Wash Ni-NTA column with 10 CV (50mL) high salt buffer and 10 CV (50mL) wash buffer.

Elute His 6 -ubiquitin-TauRD with 20mL elution buffer and collect everything.

Dilute eluted protein 1:5 with PIPES buffer to reduce the amount of salt (20mL eluted protein + 80mL PIPES buffer).

Incubate diluted His 6 -ubiquitin-TauRD protein with 0.5mg Usp2 ubiquitin protease at 4°C``1h 0m 0s.

Load the cleavage mixture onto a Source S cation exchange column previously equilibrated with cation exchange buffer A.

Wash the column with 5 CV of cation exchange Buffer A.

Elute TauRD with a 0-500millimolar (mM) linear NaCl gradient in 50millimolar (mM) PIPES-NaOH 6.5, 2millimolar (mM) β-ME (0-50% gradient from cation exchange buffer A to cation exchange buffer B over 10 CV).

Analyze eluted fraction by SDS-PAGE and Coomassie blue staining.

Load TauRD-containing fractions onto a Superdex-75 column previously equilibrated with PBS.

Analyze eluted fraction by SDS-PAGE and Coomassie blue staining.

Pool fractions containing TauRD, aliquot and flash-freeze in liquid nitrogen for storage at -80°C.