Sephacryl s 200

A 20 mL sample of M. sexta plasma collected 24 h after injection of larvae with S. cerevisiae as described above was subjected to size exclusion chromatography on a column (120 × 2.5 cm) of Sephacryl S-200 (GE healthcare) and eluted with 20 mM Tris, 150 mM NaCl, pH 7.5 at 2 mL/min, with collection of 8 mL fractions. Protein content of the fractions was assessed by absorbance at 280 nm. Samples of each fraction (100 μL) were assayed for anti-yeast activity as described above.

Human TPI enzyme was purified as outlined previously [3 (link)]. Briefly, the coding sequence for H. sapiens TPI was cloned into the bacterial expression vector pLC3 using standard techniques. The resulting plasmid directs expression of TPI containing N-terminal His₆- and MBP tags, both of which can be removed with TEV protease. TPI protein was expressed in BL21(DE3) Codon-Plus (RILP) E. coli (Agilent Technologies) grown in ZY auto-induction media (Studier, 2005) at room temperature for 24–30 hours. Cells were harvested by centrifugation, lysed via homogenization in 25 mM Tris pH 8.0, 500 mM NaCl, 10% glycerol, 5 mM imidazole, 1 mM β-mercaptoethanol and cleared by centrifugation at 30,000 × g. TPI was purified by nickel affinity chromatography followed by overnight TEV protease treatment to cleave the His₆-MBP tag from TPI. A second round of nickel affinity purification was performed to separate the His₆-MBP and TEV protease. TPI protein was further purified using anion-exchange chromatography (HiTrap-Q) followed by gel filtration (Sephacryl S-200, GE Healthcare). Peak fractions were concentrated to 4–8 mg/ml in 20 mM Tris pH 8.8, 25 mM NaCl, 2.0% glycerol and 1 mM β-mercaptoethanol using a Vivaspin concentrator (GE Healthcare). The purity was >99% as verified by SDS-PAGE.

B. viridis strain DSM 133 cells were grown anaerobically in 1:1 YPS/RCV media and harvested^{25 (link)}. A Branson 450 sonifier (Fisher Scientific, Hampton, NH) was used to break the cells. After sonication, the sample was centrifuged at 10 000 × g for 1 h at 4 °C using a Sorvall SS-34 rotor (Thermo Fisher, Waltham, MA) to pellet the cell debris. The supernatant was spun at 450 000 × rpm for 4 h at 4 °C using a Beckman Type 70 Ti (BT) rotor (Beckman Coulter, Brea, CA). After pelleting the membranes, 30% (v/v) lauryldimethylamine N-oxide (LDAO, Sigma, St Louis, MO) was added to the membrane pellets resuspension. The resuspension was stirred at 1.5% (v/v) final LDAO concentration at room temperature for ~1 h followed by centrifugation (450 000 × rpm for 1 h at 4 °C using a Beckman Type 70 Ti (BT) rotor (Beckman Coulter, Brea, CA)). The supernatant was loaded on a HiTrap Q HP anion-exchange column (GE Healthcare, Chicago, IL) and eluted with a gradient of NaCl-containing buffer (20 mM Tris). The separation was further elaborated by elution from a Sephacryl S-200 (GE Healthcare, Chicago, IL) gel filtration column previously equilibrated with a buffer containing 100 mM NaCl (20 mM Tris).