Expression and purification of mCherry-NDP52

pETDuet-1_6xHis-TEV-mCh-NDP52 (Addgene ID: 187829) was transformed into E. coli Rosetta pLySS cells.

To express the protein grow E. coli Rosetta pLySS cells in 4 L of LB medium (w Amp/Cam) at 37°C until an OD_{600 nm} of 0.4. Next, bring the temperature down to 18°C and grow further to an OD_{600 nm} of 0.8. Induce protein expression with 0.5 mM IPTG and grow for further 16 h at 18°C.

Pellet the cells at 4000 rpm 4°C 0h 15m 0s . Re-suspended the cell pellet in a buffer containing 50mM HEPES pH7.5, 300 mM NaCl, 1 mM MgCl₂, 10 mM Imidazole, 2 mM Beta-Mercaptoethanol, cOmplete protease inhibitors (Roche), and DNase (Sigma). Snap freeze in liquid nitrogen and store in -80°C until the day of purification.

Open the cells by thawing in RT water bath and sonicating 3 x 30 seconds at 50% power using a Bandelin sonicator.

Clear the lysate by ultracentrifugation (40,000 rpm for 45 min at 4°C in a Ti45 rotor, Beckman).

Filter the supernatant with a 0.45 μm syringe filter on ice.

Apply to a 5-ml His-Trap column (Cytiva) and elute with a stepwise imidazole gradient (50, 75, 100, 150, 200, and 300 mM). Fractions at 75–100 mM imidazole should contain His-TEV-mCh-NDP52. Pool those fractions and subject to TEV protease cleavage over night at 4°C by very gentle rolling.

After TEV cleavage, concentrate the protein using a 50kDa cut-off Amicon filter to 0.5 ml and inject onto a Superdex 200 increase 16/600 column (Cytiva) pre-equillibrated with a buffer containing 25mM HEPES pH7.5, 150mM NaCl, 1mM DTT at 4°C.

Pool fractions containing pure protein, concentrate using a 50kDa cut-off Amicon filter, snap freeze in liquid nitrogen, and store at −80°C.