EXPRESSION AND PURIFICATION OF HUMAN NEMO (GST-GFP-NEMO)

Grow E. coli Rosetta (DE3) pLysS cells in 2L LB medium at 37°C until achieving an OD_{600 nm}of 0.4.

Reduce temperature to 18°C and continue growth to OD_{600 nm}of 0.8.

Induce protein expression with 250micromolar (µM) IPTG and continue growth for ~16h 0m 0s at 18°C

Centrifuge the cells at 3000 rcf (4°C ,0h 15m 0s )

Aspirate media and resuspend cell pellet in ~ 10 ml of 1X PBS, centrifuge the cells at 3000 rcf (4°C ,0h 15m 0s ) and take off the supernatant.

Flash freeze the pellet in liquid nitrogen and store at -80°C until use.

Thaw pellet and resuspend in Lysis Buffer with freshly added 2millimolar (mM) β-Mercaptoethanol, Roche Protease Inhibitor and DNAse I

Sonicate sample 0h 0m 30s at 65% power for 5 cycles (Bandelin Sonopuls)

Repeat sonication for a total of 3X

Centrifuge at 48000 rcf at 4°C for 0h 45m 0s

During this step, equilibrate 3mL Glutathione Sepharose 4B beads by washing the slurry with water and Wash Buffer 1 (~ 6 ml).

Filter supernatant after centrifugation through a 0.45 µm syringe filter

Incubate the sample on equilibrated beads on a tube roller at 4°C for 4h 0m 0s

Wash beads 5X with Wash Buffer 1

Wash beads 1X with Wash Buffer 2

Wash beads 2X with Wash Buffer 1

Cleave the resulting sample to produce GFP-NEMO by incubating with thrombin 4h 0m 0s at 4°C

Centrifuge beads at 3000 rcf for 0h 3m 0s at 4°C

Collect supernatant and filter through .45 µm syringe filter

Concentrate product to a final volume of 500µL using a 10 kDa MWCO concentrator

The concentrated and filtered protein can be applied onto a Superose 6 increase column (10/300 Cytiva) pre-equillibrated with SEC buffer

Fractions containing the purified proteins are pooled, concentrated, frozen in liquid nitrogen and stored at - 80°C