Expression and purification of recombinant proteins in bacteria.

Expression vectors for GST-RNF181 and GST-RNF181 R118A were made by cloning the respective RNF181 and RNF181 R118A cDNAs into pGEX6P to fuse the proteins to the C terminus of glutathione S-transferase (GST). Proteins were expressed in BL21 bacteria (catalog number 70235-3; EMD Biosciences) as follows. Bacteria were inoculated into 250 ml LB-Amp and grown at 37°C until the optical density at 600 nm (OD₆₀₀) reached 0.75, and then IPTG (isopropyl-β-d-thiogalactopyranoside) was added to 1 mM to induce protein expression for 3 h. The cultures were cooled in an ice water bath and spun down at 7,700 × g for 10 min at 4°C. The pellets were resuspended in 12.5 ml cold phosphate-buffered saline (PBS) containing lysozyme at 0.5 mg/ml, and the mixture was incubated at room temperature for 30 min. The cultures were sonicated 10 times with 5-s pulses at 40% amplitude using a Branson 450 sonifier. Triton X-100 was added to 1%, and the lysates were centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was rotated with 400 μl glutathione-Sepharose beads (catalog number 17-5132-01; GE Healthcare) for 1 h at 4°C. The beads were spun down at 500 × g for 1 min at 4°C and washed 3 times in 8 ml of 1× PBS (pH 7.4). The beads were then loaded on a column (catalog number 731-1550; Bio-Rad), and bound proteins were eluted in 50 mM Tris HCl, pH 7.5, 10 mM glutathione elution buffer.

To express maltose-bind protein (MBP)–Bcl10, murine Bcl10 cDNA was cloned into pMALC5x to fuse Bcl10 to the C terminus of MBP. MBP-Bcl10 and MBP were expressed in BL21 cells and purified as described above for the GST fusions, except that amylose resin beads (catalog number E80215; NEB) were used, and the proteins were eluted in 50 mM Tris HCl, pH 7.5, 10 mM maltose elution buffer.

To express the myc-tagged CARD–LATCH–coiled-coil (myc-CARD-L-CC) protein, a cDNA encoding these domains was cloned into pET24a(+) in frame with N-terminal V5 and myc tags and a C-terminal 6×His tag. The protein was expressed in Rosetta cells (catalog number 71397-3; EMD Biosciences) as follows. Bacteria were inoculated into 250 ml LB with chloramphenicol and kanamycin and grown with shaking at 30°C until the OD₆₀₀ reached 0.75. IPTG at a final concentration of 1 mM was added to induce expression, and the cells were grown for 2 h at 30°C. The cultures were spun down for 15 min at 6,000 × g and 4°C and resuspended in washing buffer (20 mM Na₂H₂PO₄, 25 mM NaCl) containing 0.5 mg/ml lysozyme. Samples were freeze-thawed in dry ice, incubated at room temperature for 20 min, and then sonicated 6 times for 5 s each time at 40% amplitude. The cultures were then spun down for 15 min at 6,000 × g at 4°C, and a 1-ml bead volume of Talon resin (catalog number 635501; Clontech) was added to the supernatant, followed by rotation at 4°C for 1 h. Samples were spun down at 500 × g for 1 min at 4°C and washed 3 times in 10 ml of washing buffer. The beads were loaded on a column and eluted in elution buffer (250 mM imidazole, 500 mM NaCl, 20 mM Na₂H₂PO₄).