Hitrap sp ff column

Proteins were expressed according to a previously published protocol (41 (link)). Bacteria were lysed using a Stansted Fluid Power pressure cell homogenizer (100 MPa). Proteins harboring His tags were purified by nickel affinity chromatography as described elsewhere (29 (link)). Proteins without His tags were purified by cation exchange chromatography (CIEX) using a 5-ml HiTrap SP-FF column on a fast protein liquid chromatography (FPLC) device (Äkta purifier; GE Healthcare Life Sciences). Proteins used for the in vivo experiments were expressed in E. coli ClearColi BL21(DE3) as described elsewhere (41 (link)). In this case, protein purification was performed in an endotoxin-free environment. The purification columns and chromatography system were decontaminated using 1 M NaOH prior to sample loading. Additional purification by size exclusion chromatography (SEC) was performed following the CIEX step (Superdex 200 Increase 10/300 GL; GE Healthcare) using SEC running buffer (50 mM Na₂HPO₄, 500 mM NaCl, pH 7.4). Proteins were tested for endotoxin content using an EndoZyme kit (Hyglos, Regensburg, Germany) according to the manufacturer’s instructions. Protein identity and purity were confirmed by SDS-PAGE, followed by Coomassie staining (InstantBlue; Sigma).

Stationary phase P. acnes isolate KPA171202 were collected by centrifugation at 3600 g for 10 min. The culture supernatant was sterile filtered (0.22 μm) and precipitated with 50% ammonium sulfate. The pellet was resolved in 10 mM Tris-HCl pH 8.8 and dialyzed overnight (MWCO 3,500). The sample was loaded on an equilibrated HiTrap Q FF column (GE Healthcare, Uppsala, Sweden), and eluted with 50 mM NaCl (10 mM Tris-HCl pH 8.8). A second dialysis was performed (50 mM NaOAc-HOAc pH 5.0), samples were run on a HiTrap SP FF column (GE Healthcare), and eluted with 100 mM NaCl (50 mM NaOAc-HOAc pH 5.0). Finally, fractions were run through a 50 kDa MWCO-filter (Amicon Ultra, Ultracel-50K). The homogeneity of RoxP was verified using SDS-PAGE gels.

Synthetic DNA encoding target proteins were inserted into the pET 24 plasmid for expression in E. coli BL21. “PADRE-Trx” derived proteins contain dual 6xHis-tag and nickel affinity chromatography was applied for purification of these antigens. “OVX313-Trx” recombinant proteins were purified by cation exchange chromatography (HiTrap SP FF column, GE Healthcare) based on an arginine-rich motif at the C-terminus of the OVX313 heptamerization domain (OligoDOM technology, OSIVAX). The concentration and purity of the proteins were analyzed by SDS-PAGE–Coomassie blue staining. For endotoxin removal, all proteins were detoxified twice by Triton X-114 separation before immunization.

Recombinant oANG was expressed in CHO cells and purified as described previously [8 (link), 24 (link)]. About 30 days were required for the CHO cell-based expression of oANG. Approximately 0.8 L of serum-free culture medium was collected. Ammonium sulfate was added to the cleared culture medium to 70 % saturation. After removing most of the supernatant, the precipitate was collected by centrifugation (10,000 × g, 10 min, 4 °C). The precipitate was dissolved in 20 mM sodium acetate (pH 5.0) and dialyzed against the same buffer. The dialysate was centrifuged at 10,000 × g for 10 min at 4 °C to remove the insoluble material. The clear supernatant was loaded onto a HiTrap SP FF column (GE Healthcare) equilibrated with 20 mM sodium acetate (pH 5.0). oANG was eluted with a linear gradient of NaCl (0–1.0 M) in the same buffer. The bound fractions were pooled, concentrated using a Vivaspin Turbo 15, and subjected to gel filtration on a HiLoad 16/60 Superdex 200 pg column (GE Healthcare) equilibrated with 2 mM HEPES and 0.2 M KCl (pH 8.0). The oANG-containing fractions were pooled and concentrated to 3.0 mg/mL using a Vivaspin Turbo 15. The above molecular extinction coefficient at 280 nm was used to estimate the molar concentration of oANG.

RFC was co-expressed and purified from an E. coli BL21(DE3) Rosetta strain (pXZ2) harboring RFC1-5 subunits as previously described^{70 (link)} with modifications. Overexpression of RFC subunits was induced by 0.5 mM of ITPG for 16 h at 15 °C when cell density reached an OD₆₀₀ of 0.5. The cell pellet was suspended in buffer G (150 mM NaCl, 25 mM HEPES pH 7.6, 10% glycerol, 2 mM β-ME) supplemented with 1xPI and 1 mM PMSF. The cell lysate was prepared with a high-pressure homogenizer. The supernatant was clarified by centrifugation at 32,300×g at 4 °C for 1 h and then loaded onto a 1-ml HiTrap SP FF column (GE Healthcare, 17-5054-01) equilibrated with buffer G. Elution was performed with a continuous gradient of NaCl from 150 to 600 mM. The peak fractions containing RFC were pooled and dialyzed in buffer H (300 mM NaCl, 25 mM HEPES pH 7.6, 10% glycerol, 2 mM β-ME). Afterward, the protein sample was further processed with a Mono Q 5/50 GL equilibrated with buffer H. The flow-through fractions were pooled, concentrated, and applied to a Superdex 200 column equilibrated in Buffer H. Final peak fractions were pooled, aliquoted, and stored at −80 °C.