Fig. 3
From: Thermostable exoshells fold and stabilize recombinant proteins
Effect of tES on the stability of the encapsulated POI and the release of the POI with mild pH titration. a POI is stable in the dialysis buffer with and without encapsulation. The presence of tES enhanced the stability of tES-POI in b 0.4% trypsin, c 20% methanol, d 8 M urea, and e 30% acetonitrile. f tES(+)F116H/tES-POI is resistant to 15 min of thermal denaturation. g tES(+)F116H/tES-rLuc is highly resistant to repeated thermocycling (80 °C × 5 min × 10 cycles) showing a three order-of-magnitude higher activity compared with rLuc. h Recovery of active proteins from the tES. tES(+)F116H/tES-GFPuv, tES(+)F116H/tES-HRPc, and tES(+)F116H/tES-rLuc were subjected to pH 5.8 which resulted in the cage break and release of tES-POI. The released tES-POI (MW of tES-GFP [46 kDa], tES-HRPc [53 kDa], and tES-rLuc [55 kDa]) was proteolyzed by FXa/TEV to separate GFPuv (27 kDa), HRPc (34 kDa), and rLuc (36 kDa) from the tES subunit (19 kDa). The separated POIs were detected on the western blot using c-Myc antibody targeted to the c-Myc epitope downstream of POIs. i–k Size-exclusion chromatography of tES(+)F116H/tES-GFPuv, tES(+)F116H/tES-HRPc, and tES(+)F116H/tES-rLuc at pH values of 8.0 (upper panels) and 5.8 (lower panels). Each fraction was analyzed for GFPuv, HRPc, and rLuc activity and the data were overlaid on the respective chromatogram. All experiments were performed in triplicates and error bars represent means ± standard deviation (SD)
