LFn fusion protein preparation

In our Science Advances article, we utilized LFn-Flagellin combined with protective antigen (PA) as a tool to activate NLRC4. The LFn domain forms a complex with PA, enabling the translocation of LFn-Flagellin into cells and triggering inflammasome activation. This method was originally established by Feng Shao (doi:10.1038/nature10510). Furthermore, the use of the PA/LFn system to deliver target proteins into cells can be traced back to an earlier work (doi:10.1111/j.1365-2958.1995.tb02375.x).

Below, we provide our protocol for the expression and purification of the LFn-Flagellin protein, which we hope will be helpful for the readers.

1. Transform the pET15b LFn-Fla (C-terminal 6×His tag) plasmid (Plasmid #84871, Addgene) into Rosetta2 competent cells. Plate the transformed cells on an LB agar plate containing ampicillin (100 μg/mL, Ampicillin sodium salt, Sangon, AB0028) and incubate overnight at 37°C. 
2. Pick a single colony from the plate and inoculate it into 15 mL of LB medium (LB recipe: 8 g tryptone, 8 g NaCl, and 6.4 g yeast extract per 800 mL) containing ampicillin (Final concentration: 100 μg/mL). Culture the bacteria in a shaker at 220 rpm, 37°C, for ~8 hours. 
3. Prepare 800 mL of LB medium in 2 L Erlenmeyer flasks. Autoclave the medium in advance and supplement it with ampicillin. Add 6 mL of the bacterial culture from step 2 into each flask and culture in a shaker at 220 rpm, 37°C, for 2–3 hours until the OD₆₀₀ reaches ~2.
4. Induce protein expression by adding IPTG (BBI, IB0168) to a final concentration of 0.4 mM. Continue to culture bacteria overnight in a shaker at 220 rpm, 30°C.
5. Centrifuge the culture at 4200 rpm for 15 minutes to pellet the cells. 
6. Discard the supernatant and resuspend the pellet thoroughly in pre-cooled Lysis Buffer (50 mL per 800 mL culture, Lysis Buffer recipe: 50 mM Tris-HCl (pH 8.0), 250 mM NaCl, and 20 mM Imidazole).
7. Add Triton X-100 (Sangon, A110694-0100) to the solution to a final concentration of 1%. Add Protease Inhibitor Cocktail (MCE, HY-K0010) to the solution to 1×. 
8. Use a cell disruptor to lyse the bacteria following the manufacturer’s instructions.
9. Centrifuge the lysate at 20,000×g for 25 minutes at 4°C. Collect the supernatant. For every 50 mL of the supernatant, use 0.5 mL of His-tag resin beads (Ni-NTA Resin, MCLAB, NINTA-100).
10. Equilibrate the beads by softly centrifuging at 500 × g for 30 seconds at 4°C, discarding the supernatant, and resuspending in 1 mL of Lysis Buffer per 0.5 mL of beads. Mix for 3–5 minutes on a vertical rotator. Repeat this step for 3 times.
11. Incubate the equilibrated beads with the lysate on a vertical rotator at 4°C overnight.
12. Centrifuge at 500 × g for 60 seconds at 4°C, discard the supernatant, and resuspend the beads in 50 mL of Lysis Buffer. Wash for 5 minutes at 4°C on a vertical rotator.
13. Repeat the wash step twice. After the final wash, transfer the beads to 1.5 mL Eppendorf tubes (up to 0.5 mL of the resin per tube). Perform additional 2–3 washes with 1 mL of Lysis Buffer per tube.
14. Add 1 mL of Elution Buffer (25 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 400 mM Imidazole) to the beads and incubate on a vertical rotator at 4°C for 30 minutes.
15. Centrifuge at 500 × g for 60 seconds at 4°C and collect the supernatant into a new 1.5-mL tube.
16. Repeat the elution step once. Store each elution fraction in separate tubes.
17. Add the elution solution to a 50 kDa ultrafiltration tube (Millipore Amicon Ultra-15, UFC905008). Centrifuge at 3700 × g for 10 minutes at 4°C, discard the flow-through, and dissolve the protein (retained on the filter membrane) in 5 mL of PBS.
18. Repeat the centrifugation and dissolution process twice. Store the concentrated protein solution at -80°C. 
19. Analyze the purified protein by SDS-PAGE with Coomassie Brilliant Blue staining. Include serially diluted BSA as standards (3 mg/mL, 2 mg/mL, 1 mg/mL, and 0.5 mg/mL) to estimate the protein concentration and purity.