NDP52 and OPTN S177D S473D: expression and purification

Human NDP52 and human OPTN S177D S473D genes were cloned into bacterial expression vector. Addgene IDs: 187828 and 187827, respectively.

The proteins were expressed in E. coli Rosetta pLySS cells. Grow the cells in 4 L of LB medium 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 100 μM 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 lysis buffer containing 50 mM HEPES, pH 7.5, 300 mM NaCl, 2 mM MgCl2, 2 mM β-mercaptoethanol, cOmplete protease inhibitors (Roche), DNase and flash freeze in liquid nitrogen. Store in -80°C until the day of purification.

Open the cells by thawing and sonicating 2 x 30 seconds.

Clear the lysate by centrifugation (25 000 rpm for 30 min at 4°C in a Ti45 rotor, Beckman).

Incubate the cleared supernatant with 5 ml of Glutathione Sepharose 4B beads slurry (Cytiva) for 1h at 4°C rolling slowly. The beads slurry should be washed with water and then with 50 mM HEPES, pH 7.5, 300 mM NaCl, and 1 mM DTT beforehand.

After 1h of incubation with the cleared lysate wash the beads four times with 40 ml low salt buffer (50 mM HEPES, pH 7.5, 300 mM NaCl, and 1 mM DTT) buffer, followed by one wash with high salt buffer (50 mM HEPES, pH 7.5, 700 mM NaCl, and 1 mM DTT) and again two washes with low salt buffer.

Finally, incubate 0h 15m 0swith TEV protease at 4°C. 20 ul of 10 mg/ml home-madeTEV protease.

The next day spin down the beads (4000 rpm, 3 min, 4°C) and collect the supernatant containing cleaved protein.

Filter the supernatant through a 0.45 μm syringe filter to remove any residual beads.

Concentrate the protein down to 0.5 ml using 30kDa cut-off Amicon filter and apply onto a Superose 6 increase column (10/300 Cytiva) pre-equillibrated with a buffer containing 25 mM HEPES, pH 7.5, 150 mM NaCl, and 1 mM DTT.

Pool fractions containing pure proteins (see attached pdf), concentrate, snap freeze in liquid nitrogen, and store at −80°C.