Purification of Truncated Staphylococcus aureus Sortase A

The 59 amino acid truncated mutant of Staphylococcus aureus sortase A (Sa-SrtAΔ59)^{29 (link)} was polymerasechain reaction (PCR) amplified from pET28a (Addgene plasmid no. 51138) to append a N-terminal His₆ tag and NdeI and EcoRI sites for ligation into the pRSET-A vector (Invitrogen). This eliminated the thrombin cleavage site separating the His₆ tag from the SrtA protein in the original vector. The construct was transformed into competent Escherichia coli Shuffle cells (NEB). A fresh bacterial stab was inoculated into 500 mL of ZYP-5052 autoinduction media (Amresco, Solon, OH) containing ampicillin (100 μg/mL) and antifoam 204 (Sigma-Aldrich, Saint Louis, MO) and grown for 3–4 days at room temperature (RT). Following centrifugation, bacteria were lysed using a solution of B-PER (Thermo Fisher Scientific), 0.4 mg/mL lysozyme (Amresco), 0.4 μg/mL DNase (Sigma), and Complete Mini-EDTA-free protease inhibitor tablets (Sigma). Lysates were freeze–thawed, and the supernatant was purified using Ni-NTA agarose (Qiagen, Germantown, MD) per manufacturer protocol.