Table 4 Preparation of recombinant CobB TIMING: 2 days

 1. Clone the Salmonella enterica deacetylase CobB gene (Supplementary Information 1 and 2) into the plasmid pET22b(+)-His.

 2. Introduce the constructed expression plasmid into E. coli BL21 (DE3) cells with standard CaCl₂ transformation.

 3. Grown the transformed cells at 37 °C in LB medium containing 50 μg/mL ampicillin.

 4. Add IPTG to a final concentration of 1.0 mM when the optical density at 600 nm of the culture reaches 0.6–0.8.

 5. Keep the IPTG-induced cells growing for 6 h. Then, harvest the cells by centrifugation at 800×g for 5 min.

 ■PAUSE POINT The harvested cells can be stored at − 20 °C for weeks before further processing.

 6. Disrupt the cells using an ultra-high pressure continuous flow cell disrupter in ice-cold 1× PBS containing 1 mM PMSF and 0.5 mM DTT.

 ! CAUTION PMSF is highly toxic; handle with caution.

 7. Remove cell debris by centrifugation at 21,000×g for 40 min at 4 °C.

 8. Load the supernatant (10 mL) onto a Nickel resin column, and process the column with the AKTA™ FPLC™ System.

 9. Wash the bound protein with 10 volumes of binding buffer, and elute the proteins using elution buffer.

 10. Desalt the proteins using gel chromatography with desalination buffer.

 11. Check the purity of the proteins using SDS-PAGE before storing at − 80 °C.