Pet22b vector

E. coli BL21 and E. coli BL21 (DE3) were used. Genes for N. meningitidis CSS (UniProtKB ‐ Q7DDU0) and SiaC (UniProtKB ‐ P0A0Z8) were from Galab Laboratories. Both were cloned (Online Supporting Information) into a pC21e1 expression vector reported recently (Zhong et al., 2019 (link)). The NAL gene from L. plantarum WCFS1 (GenBank CCC80530.1) was codon‐optimized for expression in E. coli and received in a pET22b (+) vector (GenScript Biotech). The gene for ManNAc 1‐dehydrogenase (ManNAcDH, E.C. 1.1.1.233) from Flavobacterium sp. 141‐8 was kindly provided in a pET‐28a(+)‐vector by Kathrin Castiglione (Friedrich‐Alexander Universität Erlangen‐Nürnberg, Germany) (Klermund et al., 2016 (link)). E. coli strains were cultured in LB broth and agar plates.

Synthesized DNA fragments (SP_GRP-CBM11-IBP-His₆ and SP_GRP-CBM11-4xIBP-His₆) were cloned into a pET-22b (+) vector (Genscript, Piscataway, NJ) and transformed into Escherichia coli BL21 for protein expression. Starter cultures of each expression strain were inoculated into one liter of LB broth containing the appropriate antibiotic and grown at 37 °C until OD₆₀₀ = 0.4. Cultures were induced with 0.25 mM IPTG and grown overnight at 17 °C.
The lysis of frozen cell pellets was conducted as described in Chung et al. except at room temperature [107 (link)]. The cell mixture was sonicated at room temperature for two min using a Branson 5510 water bath sonicator (Branson Ultrasonics Corporation, Danbury, CT). Centrifugation at 15,000×g for 20 min was performed to remove cell debris. The resulting supernatant in buffer A (50 mM Tris pH 8.0, 100 mM NaCl, 10 mM imidazole) was loaded onto a 5 mL HisTrap FF crude column (GE Healthcare, Piscataway, New Jersey, USA) on an AKTA FPLC (GE Healthcare, Piscataway, New Jersey, USA) and washed with buffer A. After washing, the protein was eluted with buffer B (50 mM Tris pH 8.0, 100 mM NaCl, and 200 mM imidazole). Affinity-purified proteins were further purified by size-exclusion chromatography using a HiLoad 16/600 Superdex 75 pg column (GE Healthcare, Piscataway, New Jersey, USA) in buffer C (50 mM Phosphate pH 7.0).

The mature porcine IL-18 nucleotide sequence (NCBI: NM_213997.1) was designed and linked to 6 histidine residues (6His-tag) at the N-terminal and cloned into the pET22b (+) vector (GenScript, Hong Kong, China). Protein expression was induced in a 5 L culture of E. coli BL-21 (DE3) strain transformed with the construct pET22b-pIL-18, with culture conditions of 37 °C, pH 7.0, and 0.5 mM isopropyl β-D1-thiogalactopyranoside (IPTG), for 5 h. Cells were harvested at 4260× g for 10 min at 4 °C. Cells were resuspended in NaH₂PO₄ buffer (50 mM), NaCl (300 mM), imidazole (3 mM), and urea (8 M). The solution was lysed by mechanical disruption in a high-pressure homogenizer (EmulsiFlex C-5, Avestin, Inc., Ottawa, ON, Canada). The sample was centrifuged at 17,500× g for 10 min at 4 °C for the soluble fraction recovery. A negative expression control culture was performed in the same conditions, using an empty pET22b (+) vector. The protein expressed in this system will be called epIL-18.