Publication protocol
"For the expression of each clone, a single positive colony was selected from a freshly transformed plate and grown in 100-mL LB broth, with kanamycin (50 µg/mL, for a protein gene on pRSF1b vector), ampicillin (100 µg/mL, for a protein gene on pBAD/HisB vector), or both (cotransformation) used as selection markers. Following overnight incubation at 37 °C, 12.5 mL of the starter culture was used to inoculate a 500-mL LB broth and allowed to grow until an absorbance (OD600) of 0.4–0.5 was reached. Protein expression was then induced with 0.4 mM IPTG (pRSF vector), 0.1% L-arabinose (pBAD vector), or both (cotransformation). We tested the role of a tightly controlled relative expression of the encapsulated POI by evaluating the effect of L-arabinose (0.01, 0.1, and 1%) on the functional expression of GFPuv. After 4 h of incubation at 37 °C, cells were pelleted by centrifugation at 13,750 ×g for 10 min. The cell pellet was resuspended in a lysis buffer (25 mM Tris-HCl, 150 mM NaCl, pH 7.5), sonicated, and centrifuged to separate cell debris. The supernatant obtained was purified using a two-step chromatography procedure. pRSF clones were subjected to hydrophobic interaction chromatography (HIC) using HiPrepTM Phenyl FF (low sub) 16/10 (GE healthcare), followed by SEC using a Superdex S-200 10/300 GL column (GE healthcare). pBAD clones were subjected to Ni-NTA (Expedeon) column chromatography, followed by SEC. Fusion proteins were purified using Ni-NTA and SEC. The purity of the SEC fraction was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Buffers used were as follows: HIC: buffer A, 25 mM Tris-HCl, 150 mM NaCl, and 1 M (NH4)2SO4, pH 7.5; buffer B, 25 mM Tris-HCl, 150 mM NaCl, pH 7.5; Ni-NTA chromatography: buffer A, 25 mM Tris-HCl, 150 mM NaCl, pH 7.5; buffer B, 25 mM Tris-HCl, 150 mM NaCl, and 500 mM imidazole, pH 7.5; and SEC: 25 mM Tris-HCl, pH 8. All buffers used for the lysis, purification, and assays of HRPc clones were supplemented with 5 mM CaCl2 and 2.5 µM hemin (a stock solution of 40 mM was prepared by dissolving 25 mg of hemin in 1 mL of 1.4 N ammonium hydroxide).
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