Expression and purification of SrtBΔ30 and its mutants

The DNA sequence of SrtB_Δ30 was amplified from S. aureus 29213 genomic DNA as a template and was cloned into pET-28a. After that, the plasmid was transfected into E. coli BL21 (DE3) for expression.

The mutants of SrtB_Δ30 (N92A, Y128A) were produced from the pET-28a plasmid using the Quick Change site-directed mutation kit (Stratagene, La Jolla, CA, USA). After digestion with the DpnI enzyme, the plasmids were transfected into E. coli BL21 (DE3). The primer pairs used in this study are presented in Table S1.

E. coli was cultured in the LuriaBertani (LB) medium. Isopropyl-β-d-thiogalactoside (IPTG) was added when the optical density at 600 nm (OD₆₀₀) reached 0.8. The bacteria were pelleted by centrifugation and lysed ultrasonically. The supernatant was loaded onto a His-affinity column (GE Healthcare Life Sciences). The contaminant proteins were eliminated with 10 mM imidazole in an equilibration buffer (300 mM NaCl, 50 mM NaH₂PO₄, 10 mM Tris-HCl, pH 8.0). SrtB_Δ30 and the mutant proteins were eluted with 200 mM imidazole in an equilibration buffer.