HeV NCORE pET43.1a

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	By inoculating a single colony into 10 mL 2× YT media containing ampicillin (100 μg/mL) and glucose (2.0%) overnight at 37 °C, with shaking at 160 rpm. The overnight cultures were used to inoculate 10 mL fresh 2× YT media (HeV NFL) or Terrific Broth (HeV NCORE) containing ampicillin (100 μg/mL) and glucose (0.1%) at a starting OD600nm of 0.1 and the culture was grown at 37 °C, 160 rpm, until an OD600nm of 0.5 was reached, at which point the temperature was reduced to 26 °C for HeV NFL and 18 °C for HeV NCORE. At an OD600nm of 0.8, the expression of both HeV N constructs was induced by the addition of 1.0 mM isopropyl-β-d-thiogalactopyranoside (IPTG, GoldBio) and 0.25% arabinose (Sigma–Aldrich) and protein expression monitored over a 20 h period by the collection of 200 μL culture aliquots at T = 0, 4, and 20 h post-induction.

Protocol tips

By inoculating a single colony into 10 mL 2× YT media containing ampicillin (100 μg/mL) and glucose (2.0%) overnight at 37 °C, with shaking at 160 rpm. The overnight cultures were used to inoculate 10 mL fresh 2× YT media (HeV NFL) or Terrific Broth (HeV NCORE) containing ampicillin (100 μg/mL) and glucose (0.1%) at a starting OD600nm of 0.1 and the culture was grown at 37 °C, 160 rpm, until an OD600nm of 0.5 was reached, at which point the temperature was reduced to 26 °C for HeV NFL and 18 °C for HeV NCORE. At an OD600nm of 0.8, the expression of both HeV N constructs was induced by the addition of 1.0 mM isopropyl-β-d-thiogalactopyranoside (IPTG, GoldBio) and 0.25% arabinose (Sigma–Aldrich) and protein expression monitored over a 20 h period by the collection of 200 μL culture aliquots at T = 0, 4, and 20 h post-induction.

Publication protocol

Small-scale recombinant protein expression studies were performed by inoculating a single colony into 10 mL 2× YT media containing ampicillin (100 μg/mL) and glucose (2.0%) overnight at 37 °C, with shaking at 160 rpm. The overnight cultures were used to inoculate 10 mL fresh 2× YT media (HeV NFL) or Terrific Broth (HeV NCORE) containing ampicillin (100 μg/mL) and glucose (0.1%) at a starting OD600nm of 0.1 and the culture was grown at 37 °C, 160 rpm, until an OD600nm of 0.5 was reached, at which point the temperature was reduced to 26 °C for HeV NFL and 18 °C for HeV NCORE. At an OD600nm of 0.8, the expression of both HeV N constructs was induced by the addition of 1.0 mM isopropyl-β-d-thiogalactopyranoside (IPTG, GoldBio) and 0.25% arabinose (Sigma–Aldrich) and protein expression monitored over a 20 h period by the collection of 200 μL culture aliquots at T = 0, 4, and 20 h post-induction. The cells were harvested by centrifugation at 14,000 rpm for 5 min and then frozen. Bacterial cell pellets were solubilized by incubation in 80 μL of His A buffer (20 mM sodium phosphate buffer, pH 7.5 containing 500 mM NaCl, and 20 mM imidazole) supplemented with 2 mM MgCl2, 0.25 mg/mL lysozyme and 25 U/mL Benzonase® (Merck Millipore) followed by three freeze–thaw cycles and incubation at 37 °C for 20 min. To separate the soluble protein fraction from the insoluble fraction, the lysate was centrifuged at 14,000 rpm for 5 min. The soluble protein supernatant was removed and the remaining insoluble pellet was resuspended in 80 μL of 8 M urea. Soluble and insoluble time-course samples were mixed with 4× LDS SB (Life Technologies) for analysis by SDS–PAGE and Western blotting.

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