Recombinant MLP Protein Expression in E. coli

The co-expression of HIS₆-TxtA^A and HIS₆-TxtB^A with HIS₆-tagged MLPs was conducted as previously described (Li et al., 2019a (link)). Briefly, the expression strain E. coli BL21(DE3)ybdZ:aac(3)IV (Table 1) containing either pACYCDuet-1/HIS₆-txtA^A or pACYCDuet-1/HIS₆-txtB^A, with and without a pET28b-derived MLP expression plasmid (Table 2), was cultured overnight in 3 mL of LB medium supplemented with 1% w/v glucose and the appropriate antibiotics. The overnight cultures were subcultured into fresh LB medium containing appropriate antibiotics, and the cultures were incubated at 37°C and 200 rpm until the OD₆₀₀ reached 0.4–0.6. The production of the HIS₆-tagged proteins was induced by adding 1 mM isopropyl β-d-thiogalactopyranoside (IPTG) and then incubating the cultures at 16°C and 200 rpm for 48 h. Cells from 1 mL of culture were harvested and were resuspended in 200 μL of 50 mM Tris–HCl (pH 8.0) containing 1 × cOmplete EDTA-free protease inhibitor. The cells were lysed by sonication and the cell debris was removed by centrifugation. The soluble proteins were collected, and the protein concentration was quantified using a Bradford protein assay kit (Fisher Scientific).