2.1. Protein expression and purification  

The expression plasmid for full-length abMurG corresponding to amino acids Met1–Met365 was constructed by inserting the synthesized gene product, digested at the NdeI and XhoI restriction sites, into pET-21a vector. The gene sequence was derived from GenBank (ID {"type":"entrez-protein","attrs":{"text":"SVJ97884","term_id":"1455605536","term_text":"SVJ97884"}}SVJ97884), and gene synthesis was conducted by BIONICS (Seoul, Republic of Korea). The expression vector containing the abMurG gene was delivered into the E. coli BL21 (DE3) strain by heat shock at 42°C. The transformed bacteria were spread on a lysogeny broth agar plate containing kanamycin and incubated at 37°C for 16 h. A single recombinant colony was selected and cultured overnight at 37°C in 5 ml lysogeny broth containing 50 µg ml⁻¹ kanamycin, following which the cells were transferred and cultured on a large scale (6 l). When the optical density at 600 nm reached approximately 0.6–0.7, 0.5 mM isopropyl β-d-1-thiogalactopyranoside was added to the medium to induce gene expression, and the cells were further cultured at 20°C for 18 h in a shaking incubator. Subsequently, the bacterial cells were harvested by centrifugation and the pellet was resuspended in 20 ml lysis buffer (20 mM Tris–HCl pH 7.9, 500 mM NaCl). After adding phenylmethylsulfonyl fluoride, a serine protease inhibitor (Sigma–Aldrich, St Louis, USA), the cells were disrupted by sonication on ice with six bursts of 30 s each and a 60 s interval between bursts. The cell lysate was centrifuged at 10 000g and 4°C for 30 min to remove cell debris. The supernatant was collected and mixed with nickel–nitrilotriacetic acid resin solution (Qiagen, Hilden, Germany) by gentle agitation at 4°C overnight. The resulting mixture was inserted into a gravity-flow column pre-equilibrated with lysis buffer. The column was washed with 200 ml washing buffer (20 mM Tris–HCl pH 7.9, 500 mM NaCl, 25 mM imidazole) to remove unbound proteins. 3 ml elution buffer (20 mM Tris–HCl pH 7.9, 500 mM NaCl, 250 mM imidazole) was then loaded into the column to elute the bound protein. The resulting eluate was concentrated to 20 mg ml⁻¹ and was subsequently subjected to size-exclusion chromatography (SEC). SEC purification was conducted using an ÄKTAexplorer system (GE Healthcare, Chicago, USA) equipped with a Superdex 200 Increase 10/300 GL 24 ml column (GE Healthcare) pre-equilibrated with SEC buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl). The SEC peak fractions were pooled, concentrated to 8 mg ml⁻¹, flash-cooled in liquid N₂ and stored at −80°C until use. Protein purity was assessed by SDS–PAGE.