Hitrap deae ff column

Lipid II was synthesized in vitro as described previously (Schneider et al., 2004 (link)). Briefly, isolated membranes from Micrococcus flavus (Schneider et al., 2004 (link)) were incubated with UDP-MurNAc-pentapeptide from Staphylococcus simulans 22 or Bacillus cereus T (Schneider et al., 2004 (link)), C₅₅-P (Larodan, Solna, Sweden) and UDP-GlcNAc (Sigma-Aldrich, Taufkirchen, Germany). Optimal conditions had to be titrated in an analytical assay before preparing lipid II in a larger scale. Lipid II was obtained by mixing 20–30 μl isolated membranes, 7.5–15 μl UDP-MurNAc-pentapeptide, 5 nmol C₅₅-P, 1 mM UDP-GlcNAc, 5 mM MgCl₂, 60 mM Tris (pH 7.5), 0.5% Triton X-100 (v/v) in a final volume of 75 μl. The mixture was incubated for 4 h at 30°C with shaking and afterwards extracted and visualized by thin layer chromatography (TLC) as described previously (Schneider et al., 2004 (link); Klöckner et al., 2014 (link)). Lipid II synthesis in a preparative scale was achieved by using a 200-fold volume of the analytical scale.
Purification of lipid II was performed on a 5 mL HiTrap DEAE FF column (GE Healthcare, Freiburg, Germany) and eluted with a linear gradient of chloroform/methanol/water (2:3:1, v/v) to chloroform/methanol/300 mM ammonium bicarbonate (2:3:1, v/v). Lipid II was quantified by measuring the released phosphate upon total hydrolysis (Rouser et al., 1970 (link)).

Salts were purchased from Sigma-Aldrich (Milan, Italy). Reagents were obtained from Mallinckrodt Baker (Milan, Italy). The Sartorius Vivaflow 200 ultrafiltration system, equipped with a 10 kDa cutoff polythersulfone membrane, was obtained from Sartorius (Florence, Italy). Pierce Concentrators were from Thermo Fisher Scientific (Milan, Italy). Anion exchange chromatography was performed on an FPLC Akta Basic equipped with a UV-Vis detector (GE-Healthcare, Milan, Italy) using a HiTrap DEAE FF column with a volume of 5 mL (GE-Healthcare, Milan, Italy). Gel filtration chromatography was conducted on an HPLC Akta Basic equipped with a UV-Vis detector (GE Healthcare, Milan, Italy) using a progel-TSK G2000 SWXL column 30 cm × 7.8 mm (Supelco, Merck KGaA, Darmstadt, Germany). Dialysis was performed using Spectra/Por 3.5 kDa MWCO membranes (Spectrum Labs, Fisher Scientific Italia, Rodano, Milan). PVDF membranes for western blotting were obtained from Millipore (Milan, Italy). The monoclonal antibody mAbKT4 was used to detect the KT [9 (link)]. The anti-mouse secondary antibody was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The ECL (enhanced chemiluminescence) system used for the immunodetection was obtained from Amersham Pharmacia Biotech (Milan, Italy).

153 bp 601 DNA (Lowary and Widom, 1998 (link)) was prepared from a 32-copy plasmid (a gift from Dr. Tom Muir’s lab), transformed into DH5 $\alpha$  Escherichia coli cells and grown overnight in LB/ampicillin media at 37°C. Following harvest by centrifugation, Giga prep plasmid purification kits (Qiagen, cat.12191) were used to purify the 32-copy plasmid. Digestion using EcoRV (0.5units/ $\mu$ g plasmid) in NEB buffer3 was carried out for 20 hr at 37°C. The resulting solution of liberated 153 bp fragments was treated with 0.3375 vol equivalents of fresh 40% PEG-600 and 0.15 vol equivalents of 5 M NaCl solution and incubated at 4°C for 1 hr to precipitate the vector backbone. Following centrifugation at 14,000 rpm for 30 min at 4°C, 2.5 vol equivalents of 100% ethanol was added to the supernatant and left at −20°C overnight to precipitate the 153 bp 601 DNA fragments. Following centrifugation at 14,000 rpm for 30 min at 4°C, the precipitated DNA pellet was washed once with cold 70% ethanol and resuspended in 10 mM Tris pH 8. The 153 bp 601 DNA fragments were further purified using a HiTrap DEAE-FF column (GE Healthcare) equilibrated in 10 mM Tris pH 8 and eluted using a linear gradient up to 1M KCl over 9 column volumes. Fractions containing DNA were pooled, concentrated and the concentration of KCl was adjusted to 2 M for subsequent NCP reconstitution.