TITLE: Purification of recombinant Tau Repeat Domain (TauRD) from Escherichia coli
AUTHORS: Patricia Yuste-Checa, F Ulrich Hartl

[DESCRIPTION]
This protocol details how to efficiently purify the recombinant Tau repeat domain from Escherichia coli.

[GUIDELINES]
NOTE : This protocol was optimized for purification of the cysteine-free TauRD (Tau residues 244-371, C291A/P301L/C322A/V337M), but in principle any Tau isoform or mutant could be purified following this method if it is tagged with a His 6 -ubiquitin tag. If cysteines are present, reducing agent should be added to buffers to avoid the formation of disulphide bonds.

[STEPS]
SECTION: TauRD expression
1. Thaw RbCl-competent Escherichia coli Bl21 cells (DE3) on ice.

SECTION: TauRD expression
2. Add 1 µL of pHUE-TauRD plasmid (His 6 -ubiquitin-TauRD) and incubate 30 min on ice.

SECTION: TauRD expression
3. Heat shock 45 s at 42 °C.

SECTION: TauRD expression
4. Incubate on ice 2 min, then add 850 µL Lysogeny broth (LB) or Super Optimal broth with Catabolite repression (SOC) medium.

SECTION: TauRD expression
5. Shake for 60 min at 37 °C.

SECTION: TauRD expression
6. Centrifuge for 5 min at 3000 x g and remove most of the supernatant.

SECTION: TauRD expression
7. Resuspend the pellet with the remaining supernatant and plate the bacteria on LB/Ampicillin agar plates and incubate 60 min at 37 °C.

SECTION: TauRD expression
8. Prepare preculture: Scrap all colonies with the scraper and inoculate 25 mL-50 mL LB/Ampicillin. Shake at 37 °C for 4-360 min.

SECTION: TauRD expression
9. Measure OD600 of the preculture and inoculate two flasks with 1 L of TB media each to an 
OD600 = 0.05.

SECTION: TauRD expression
10. Shake flasks at 37 °C until approx. OD600  = 0.5-0.8. (2 - 240 min)

SECTION: TauRD expression
11. Add isopropyl β-D-1-thiogalactopyranoside (IPTG) at final concentration of 0.4 millimolar (mM).

SECTION: TauRD expression
12. Shake flasks 240 min at 37 °C.

SECTION: TauRD expression
13. Centrifuge bacterial culture at 4000 rpm for 60 min. Discard supernatant. Cell pellets can be stored at -80 °C.

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

SECTION: Ni-NTA chromatography
15. Add 1 1524 lysozyme and incubate gently shaking for 30 min at 4 °C.

SECTION: Ni-NTA chromatography
16. Sonicate lysate on ice, 5 cycles 30 s ON, 90 s OFF.

SECTION: Ni-NTA chromatography
17. Centrifuge lysate at 40000 x g 60 min at 4 °C.

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

SECTION: Ni-NTA chromatography
19. Load lysate supernatant to Ni-NTA column.
SECTION: Ni-NTA chromatography
20. Wash Ni-NTA column with 10 CV (50 mL) high salt buffer and 10 CV (50 mL) wash buffer.

SECTION: Ni-NTA chromatography
21. Elute His 6 -ubiquitin-TauRD with 20 mL elution buffer and collect everything.

 



SECTION: His 6 -ubiquitin cleavage
22. Dilute eluted protein 1:5 with PIPES buffer to reduce the amount of salt (20 mL eluted protein + 80 mL PIPES buffer).

SECTION: His 6 -ubiquitin cleavage
23. Incubate diluted His 6 -ubiquitin-TauRD protein with 0.5 mg Usp2 ubiquitin protease at 4 °C60 min.


 



SECTION: Cation exchange chromatography
24. Load the cleavage mixture onto a Source S cation exchange column previously equilibrated with cation exchange buffer A.
SECTION: Cation exchange chromatography
25. Wash the column with 5 CV of cation exchange Buffer A.
SECTION: Cation exchange chromatography
26. Elute TauRD with a 0-500 millimolar (mM) linear NaCl gradient in 50 millimolar (mM) PIPES-NaOH pH 6.5, 2 millimolar (mM) β-ME (0-50% gradient from cation exchange buffer A to cation exchange buffer B over 10 CV).

SECTION: Cation exchange chromatography
27. Analyze eluted fraction by SDS-PAGE and Coomassie blue staining.
SECTION: Size exclusion chromatography
28. Load TauRD-containing fractions onto a Superdex-75 column previously equilibrated with PBS.
SECTION: Size exclusion chromatography
29. Analyze eluted fraction by SDS-PAGE and Coomassie blue staining.
SECTION: Size exclusion chromatography
30. Pool fractions containing TauRD, aliquot and flash-freeze in liquid nitrogen for storage at -80 °C.