Recombinant Protein Expression and Purification

Recombinant Tcnα, TcdB, TpeL, TcsL, and TcsH were expressed in Bacillus subtilis SL401 and purified as His-tagged proteins^{52 (link)}. In brief, B. subtilis cells were cultured at 37 °C till OD₆₀₀ reached 0.6 and then induced with 1 mM isopropyl-β-D-thiogalactoside at 25 °C for 20 h. The recombinant LDLR_LA-Fc with His-tag at C-terminus was expressed in the Expi293F cells. In brief, 5 × 10⁸ Expi293F cells were transfected with 750 μg of pHLsec-LDLR_LA-Fc using 1 mg/ml PEI. The supernatant was collected 4 days post-transfection and applied to purification. All above recombinant proteins were purified by Ni-affinity chromatography and size-exclusion chromatography (GE Healthcare).