Clarified rRBD supernatant samples from the transient and stable fed-batch cultures were purified for gel analysis using a Ni-NTA Spin Kit (Qiagen) under native conditions. Spin columns were equilibrated, loaded with 3 × 600 µL harvested supernatant, washed, and eluted per the manufacturer’s protocol. Samples were buffer exchanged into PBS using 10 kDa Amicon Ultra-0.5 Centrifugal Filter Units (MilliporeSigma). rRBD concentrations were measured by A280 absorbance.
Purified rRBD samples were buffer exchanged into molecular biology grade water (Fisher Scientific) using 10 kDa Amicon Ultra-0.5 Centrifugal Filter Units (MilliporeSigma). rRBD concentrations were measured by A280 absorbance. 15 µg of rRBD was then denatured and digested with PNGaseF (Promega) based on the manufacturer’s protocol. In brief, rRBD was denatured at 95 °C for 10 min in the presence of dithiothreitol (DTT; New England Biolabs) and sodium dodecyl-sulfate (SDS; Bio-Rad). PNGase F digestion with 1U/µL enzyme was performed in the presence of sodium phosphate pH 7.4 (Fisher Scientific) and Triton X-100 (MilliporeSigma) at 37 °C for 4 h.
Purified rRBD samples diluted with 2x Laemmli sample buffer (Bio-Rad) supplemented with 120 mM DTT (New England Biolabs) were boiled for 5–10 min. 1 µg of intact protein or PNGaseF digested protein was loaded into a 4–15% Mini-PROTEAN® TGX™ gel (Bio-Rad). 5 µL of Precision Plus Protein™ Unstained Protein Standards (Bio-Rad) was used as a molecular weight marker. The gel was run for 50 min at 150 V and then stained with SYPRO® Ruby (ThermoFisher). Fix and wash buffers were prepared per the manufacturer’s protocol, and a modified rapid protocol was executed for gel fixing, staining, and washing while rocking the gel on an orbital shaker. In brief, the gel was shaken in fresh fixing buffer 2 × 15 min, stained for 3 h, and shaken in fresh wash buffer 2 × 15 min. Gels were imaged on a Typhoon FLA 7000 (Cytiva) using the 473 nm laser and 580 nm emission filter.
Purified rRBD samples were buffer exchanged into molecular biology grade water (Fisher Scientific) using 10 kDa Amicon Ultra-0.5 Centrifugal Filter Units (MilliporeSigma). rRBD concentrations were measured by A280 absorbance. 15 µg of rRBD was then denatured and digested with PNGaseF (Promega) based on the manufacturer’s protocol. In brief, rRBD was denatured at 95 °C for 10 min in the presence of dithiothreitol (DTT; New England Biolabs) and sodium dodecyl-sulfate (SDS; Bio-Rad). PNGase F digestion with 1U/µL enzyme was performed in the presence of sodium phosphate pH 7.4 (Fisher Scientific) and Triton X-100 (MilliporeSigma) at 37 °C for 4 h.
Purified rRBD samples diluted with 2x Laemmli sample buffer (Bio-Rad) supplemented with 120 mM DTT (New England Biolabs) were boiled for 5–10 min. 1 µg of intact protein or PNGaseF digested protein was loaded into a 4–15% Mini-PROTEAN® TGX™ gel (Bio-Rad). 5 µL of Precision Plus Protein™ Unstained Protein Standards (Bio-Rad) was used as a molecular weight marker. The gel was run for 50 min at 150 V and then stained with SYPRO® Ruby (ThermoFisher). Fix and wash buffers were prepared per the manufacturer’s protocol, and a modified rapid protocol was executed for gel fixing, staining, and washing while rocking the gel on an orbital shaker. In brief, the gel was shaken in fresh fixing buffer 2 × 15 min, stained for 3 h, and shaken in fresh wash buffer 2 × 15 min. Gels were imaged on a Typhoon FLA 7000 (Cytiva) using the 473 nm laser and 580 nm emission filter.
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