TITLE: GFP-Clu-tails purification from Escherichia coli cells
AUTHORS: Andreas Bracher, F Ulrich Hartl

[DESCRIPTION]
This protocol details how to purify the fusion protein GFP-Clu-tails from Escherichia coli.

[STEPS]
SECTION: His6-Ubiquitin-GFP-Clu-tails expression and cell lysis
1. Express His6-Ubiquitin-GFP-Clu-tails in E. coli Bl21 (DE3) codon+RIL cells cultured in 4 L LB Medium   at 18 °C with 0.25 millimolar (mM) IPTG.

SECTION: His6-Ubiquitin-GFP-Clu-tails expression and cell lysis
2. Centrifuge culture and keep pellet.
SECTION: His6-Ubiquitin-GFP-Clu-tails expression and cell lysis
3. Re-suspend pellet in 70 mL volume of ice-chilled Binding buffer, add Complete protease inhibitor cocktail (Roche) and 1 millimolar (mM) phenylmethylsulfonyl fluoride (PMSF).

SECTION: Ni2+-chelating Sepharose metal affinity chromatography
6. Load supernatant onto a 8 mL Ni-chelating Sepharose (Cytiva 17-0575-01) column previously equilibrated with binding buffer by gravity flow at 4 °C.

SECTION: Ni2+-chelating Sepharose metal affinity chromatography
7. Wash the column with 5 CV of ice-chilled Binding buffer.
SECTION: Ni2+-chelating Sepharose metal affinity chromatography
8. Wash the column with 2 CV of ice-chilled PBS + 50 Molarity (m) imdiazole.

SECTION: Ni2+-chelating Sepharose metal affinity chromatography
9. Elute His6-Ubiquitin-GFP-Clu-tails protein with 6x 5 mL of ice-chilled Elution buffer. Collect fractions of 5 mL volume. Bright green color indicates presence of His6-Ubiquitin-GFP-Clu-tails. Store fractions on ice.

SECTION: Ni2+-chelating Sepharose metal affinity chromatography
10. Analyze eluted fractions by SDS-PAGE and Coomassie blue staining.
 

SECTION: Protease cleavage and removal of protease
11. Pool fractions containing His6-Ubiquitin-GFP-Clu-tails peak (here fractions 4 and 5). Cleave off His6-Ubiquitin with His-tagged Usp2 during 240 min on ice.

SECTION: Protease cleavage and removal of protease
12. Exchange protein buffer to Binding buffer with a HiPrep™ 26/10 Desalting column (Cytiva 17-5087-01) equilibrated with Binding buffer at 4 °C using bright green fluorescence as a marker for protein containing fractions.

SECTION: Protease cleavage and removal of protease
13. Remove uncleaved material and His-tagged Usp2 by metal affinity chromatography as above. Collect flow-through and wash fractions with bright green fluorescence.
SECTION: Protease cleavage and removal of protease
14. Concentrate to 1.5 mL volume by ultrafiltration using 10 kDa cut-off spin concentrator at 4 °C.

SECTION: Size exclusion chromatography on HiLoad 16/600 Superdex-200 (Cytiva 28-9893-35)
15. Apply concentrate on a HiLoad 16/600 Superdex-200 (Cytiva 28-9893-35) column equilibrated with PBS. Develop the column at 4 °C and collect 3 mL fractions.

SECTION: Size exclusion chromatography on HiLoad 16/600 Superdex-200 (Cytiva 28-9893-35)
16. Analyze eluted fractions by SDS-PAGE and Coomassie blue staining.
 

SECTION: Size exclusion chromatography on HiLoad 16/600 Superdex-200 (Cytiva 28-9893-35)
17. Merge fractions 16 and 17. Concentrate to 0.7 mL volume by ultrafiltration using 10 kDa cut-off spin concentrator at 4 °C, aliquot and flash-freeze purified GFP-Clu-tails in liquid nitrogen for storage at -70 °C.
 



SECTION: His6-Ubiquitin-GFP-Clu-tails expression and cell lysis
4. Lyse cells by ultrasonication in ice bath (15 cycles of 25 s ultrasonication with 95 s intermittent cooling).
SECTION: His6-Ubiquitin-GFP-Clu-tails expression and cell lysis
5. Clear lysate by centrifugation at 22000 rpm in a JA25.50 rotor at 4 °C.