Selenomethionine medium complete

SP0092^39–491 was cloned into the pOPINF vector (OPPF-UK), truncating the first 39 residues coding for the periplasmic localization signal. The native His-tag fusion protein was expressed in Escherichia coli BL21 Rosetta cells by autoinduction using Overnight Express medium (Millipore) supplemented with 1%(v/v) glycerol, while selenomethionine-labelled protein was expressed using SelenoMethionine Medium Complete (Molecular Dimensions) supplemented with 0.5 mM IPTG for induction. Cells were lysed in 0.1 M HEPES pH 7.5, 0.5 M NaCl, 0.02 M imidazole, 10%(v/v) glycerol supplemented with EDTA-free protease inhibitors (Roche) and cleared for 1 h at 100 000g. Cleared lysates were loaded onto an affinity HisTrap HP column (GE Healthcare). The fusion protein was eluted with lysis buffer supplemented with 0.2 M imidazole and, after dilution, was treated with HRV 3C protease overnight at 4°C. The mixture was loaded onto a HisTrap HP column and the cleaved protein was immediately eluted. The resulting sample was loaded onto a Superdex 200 column equilibrated with 0.02 M MES pH 6.5, 0.2 M NaCl, 2.5%(v/v) glycerol, 0.5 mM TCEP. Fractions of the two peaks observed from gel filtration were collected separately and concentrated to 170 and 154 mg ml⁻¹ for the oligomeric and monomeric states, respectively. Macromolecule-production information is summarized in Table 1 ▸.

Two liters of E. coli BL21 CodonPlus cells expressing pETDuet-1::tipC2_ΔTMD were grown at 37 °C in 2xYT broth an OD₆₀₀ of 0.6 prior to induction of protein expression with 1 mM IPTG. Following further incubation at 37 °C for 4 h, cells were harvested by centrifugation and flash frozen. Frozen cells were thawed using lysis buffer [50 mM Tris–HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole] and lysed by sonication (6 × 30 s pulses at 30% amplitude). Insoluble cellular debris was then cleared by centrifugation and the TipC2-containing supernatant was applied to a 5 mL HisTrap™ FF Ni-NTA cartridge connected to an AKTA FPLC purification system (GE Healthcare). Unbound proteins were removed by extensive washing of the column in lysis buffer, and TipC2_ΔTMD was eluted using a linear imidazole gradient to a final concentration of 400 mM. Ni-NTA purified fractions of TipC2_ΔTMD were pooled, and the protein was further purified using a 16/600 HiLoad S200 size exclusion column (GE Healthcare) run in 20 mM Tris–HCl (pH 8.0) and150 mM NaCl. Selenomethionine-incorporated TipC2_ΔTMD was expressed an purified in an identical manner except that cells were grown in SelenoMethionine Medium Complete (Molecular Dimensions), and all purification buffers contained 1 mM tris(2-carboxyethyl)phosphine.

All amino acid point mutations in PFO were generated via QuikChange mutagenesis (Stratagene) prior to sequence verification at the Laboratory for Molecular Biology and Cytometry Research at the University of Oklahoma Health Science Center. The expression and purification of PFO and its derivatives and the CDCLs were performed as previously described for PFO (39 (link)). Frozen protein aliquots were thawed and centrifuged at 20,000 × g for 10 min and assayed for concentration prior to use in experiments.
For crystallization trials, fractions containing His₆-CDCL were pooled and dialyzed into 20 mM Na citrate (pH 6.5), 150 mM NaCl at 21°C for 16 h. Protein was concentrated to 6 mg/ml and stored at −80°C. For X-ray crystallography, selenomethionine (SeMet) CDCL was expressed in E. coli BL21(DE3) cells using Molecular Dimensions SelenoMethionine Medium Complete, according to the manufacturer’s instructions. The SeMet CDCL was purified using the wild-type protocol.