Purification of S. aureus ClpC and ClpP

Recombinant proteins (ClpC, ClpP) were overexpressed in E. coli ΔclpB/placIq cells using pDS56-based expression plasmids. Cells were grown at 30°C in 3 l 2xYT (+ 100 ug/mL Ampicillin; + 50 ug/mL Spectinomycin) until mid- exponential phase (OD600 = 0.6-0.8) and expression induced with by adding IPTG to a final concentration of 0.5 mM. Growth was continued for 3-4 h and cells harvested by centrifugation, snap frozen in liquid nitrogen and stored at -20°C. Cell pellets were thawed on ice and resuspended in 20-30 mL lysis-equilibration-wash (LEW) buffer containing protease inhibitors (8 μg/mL Pepstatin A, 10 μg/mL Aprotinin, 5 μg/mL Leupeptin) and DNAse I (10 μg/mL). The cell suspension was subsequently lysed via French Pressure Cell. Lysate was cleared by centrifugation (15.000 rpm, 45 min, 4°C), the supernatant was incubated with approx. 0.8 – 1 g Ni-IDA resin (Macherey & Nagel) for 1 h at 4°C while rotating. The suspension was packed into a gravity flow column and washed with 30 mL LEW buffer. Bound protein was eluted with LEW buffer containing 250 mM imidazole. Elutions fractions of 1 mL were collected, whose protein concentrations were determined by Bradford assay. Elution fractions were analyzed by SDS-PAGE and fractions containing ClpC or ClpP were pooled and subjected to a second purification step by size exclusion chromatography (SEC). SEC was performed using the ÄKTA purifier and Superdex 200 prep grade 16/60 columns. During SEC LEW elution buffer was exchanged against MDH low salt buffer containing 5 % glycerol. Elution fractions were analyzed by SDS-PAGE and fractions displaying highest protein purity were pooled and frozen in liquid nitrogen. Aliquots were stored at -80°C.

2xYT medium

• 16 g/L tryptone
• 10 g/Lyeast extract
• 5 g/L NaCl
• pH 7.0

LEW-Puffer

50 mM NaH₂PO4, pH 8.0, 300 mM NaCl, 5 mM beta-mercaptoethanol

MDH low salt buffer

50 mM Tris pH 7.5, 25 mM KCl, 20 mM MgCl₂, 2 mM DTT