FtsZ and GpsB purification

S. aureus GpsB-His6
The His6 tag was appended to the 3’ end of the S. aureus gpsB gene (gpsB^Sa-his6) because overexpression of gpsB^Sa-his6 in B. subtilis retained toxicity, suggesting that GpsB-His6 was functional. The gpsB gene was PCR-amplified from genomic DNA harvested from S. aureus strain SH1000 (Accession# CP059180). Kanamycin concentration for plasmid maintenance and induction times were determined empirically.
Cells were grown at 37 °C until OD600 reached between 0.6 - 0.8. Protein production was induced using 0.5 mM IPTG, after which cells were grown at 37 °C for 4 h.

Buffers
Buffer A (lysis and wash 1): 50 mM sodium phosphate (pH 8.0), 500 mM NaCl, 20 mM imidazole, 1 mM EDTA, 10% Glycerol, 3 mM DTT
Wash buffer 2: Buffer A containing 50mM Imidazole
Elution buffer: Buffer A containing 250mM Imidazole
Desalting buffer: 20 mM HEPES (pH 7.5), 250 mM KCl and 1 mM DTT and 10% Glycerol.

Procedure
The induced cell pellets were harvested, resuspended in buffer A containing 1mM PMSF, and lysed by French press. The soluble fraction of the cell lysate was separated from the debris by centrifuging the lysate at 30,000 × g for 45 min. The soluble fraction was then incubated with Ni²⁺-NTA resin pre-equilibrated with buffer A for 1 h at 4 °C. The unbound material was collected, and the resin was sequentially washed with 150 ml each of wash buffer 1 and 2. GpsB-His6 was eluted from the resin using elution buffer. The eluted protein was then desalted by buffer exchange in an Amicon filter (10,000 KDa cutoff) using desalting buffer and stored in aliquots at -80 °C.

S. aureus FtsZ
The ftsZ gene was PCR-amplified from genomic DNA harvested from S. aureus strain SH1000 (Accession# CP059180). Kanamycin concentration for plasmid maintenance and induction times were determined empirically. Cells were grown at 37 °C until OD600 reached between 0.6 - 0.8. Protein production was induced using 1 mM IPTG, after which cells were grown at 30 °C for 3 h. Cells were harvested and stored at -80 °C.

Buffers
Assay Buffer: 20 mM HEPES (pH 7.5), 250 mM KCl, 5 mM MgCl2, 10% Glycerol, 1 mM DTT
Buffer B: 20 mM HEPES (pH 7.5), 50 mM KCl, 5 mM MgCl2, 10% Glycerol (no DTT)

Procedure
Cell pellet was resuspended in buffer B and lysed by French press. The IMAC column (TALON Superflow, GE Healthcare) was equilibrated using 10 column volumes of buffer B and then the cell lysate was applied onto the column at a flow rate of 0.5 ml/minute. Protein was eluted using a gradient of 10 mM – 150 mM imidazole. Peak fractions were determined by Coomassie stained SDS-PAGE gel electrophoresis. Imidazole was removed from peak fractions by buffer exchange using PD10 column using buffer B.