Quikchange xlii

The recombinant plasmid pQE-BlGGT, previously constructed in our laboratory [30 (link)], was used as the DNA template for polymerase chain reaction (PCR) based mutations. Deletion and Ala-scanning mutagenesis were carried out with a commercially available site-directed mutagenesis kit (QuikChange XL-II; Agilent Technologies, Santa Clara, CA, USA). The complementary mutagenic primer pairs for protein engineering were designed by ourselves (Table S1) and subjected to synthesis service (Mission Biotech Co., Ltd., Taipei, Taiwan). Reaction conditions for the PCR-based mutagenesis were essentially according to the suppliers’ instructions. Following amplification, DpnI-treated PCR amplicons were transformed into XL-1-Blue supercompetent cells. Plasmid DNA isolated from the individual colonies of each transformation was sequenced to confirm the desired mutation. The mutated plasmids were accordingly called pQE-BlGGT/ΔM462, pQE-BlGGT/ΔS460-M462, pQE-BlGGT/ΔS461-M462, pQE-BlGGT/ΔP464, pQE-BlGGT/P458A, pQE-BlGGT/L459A, pQE-BlGGT/S460A, pQE-BlGGT/S461A, pQE-BlGGT/M462A, and pQE-BlGGT/P464A, respectively.

yonO was cloned into the pET-28a expression vector, and YonO was expressed in E. coli T7 Express cells (New England Biolabs) with an N-terminal 6xHis-tag. To obtain YonO^3D>3N, the yonO pET-28a construct was subjected to site-directed mutagenesis using Quikchange XL II (Agilent). Cells were grown in LB at 37 °C, and protein expression was induced at OD₆₀₀ of 0.4 with 1 mM isopropyl-β-D-1-thiogalactoside (IPTG) at 18 °C for 16 h. Harvested cells were re-suspended in grinding buffer (50 mM Tris-HCl pH 7.9, 200 mM NaCl, EDTA-free protease inhibitor cocktail (Roche)) and disrupted by sonication. The lysate was clarified by centrifugation and made to 20 mM imidazole before being loaded on a His Trap HP column (GE Healthcare) pre-equilibrated with 20 mM Tris-HCl pH 7.9 and 600 mM NaCl. Protein eluted in 100 mM imidazole was bound to a HiTrap Heparin column (GE Healthcare) equilibrated in 10 mM Tris-HCl pH 7.9 and 600 mM NaCl, and eluted by a gradient increase of NaCl concentration to 1 M. Fractions containing YonO were concentrated and further purified on a Superdex 200 16/60 column equilibrated in 50 mM Tris-HCl pH 7.9 and 500 mM NaCl. Purified YonO was concentrated and dialysed overnight into storage buffer (20 mM Tris-HCl pH 7.9, 50% glycerol, 200 mM KCl, 1 mM dithiothreitol and 0.1 mM EDTA) and stored at −20 °C.