pD5H8-NS1

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	The recombinant T. thermophila strains were cultured at 5 × 105 cells/ml in Neff medium for 16 hr and treated with 15 µg/ml of cadmium chloride to induce transgene expression. T. thermophila cells were collected by centrifugation for 5 min at 1,000 g, and then resuspended in pre-chilled buffer A (40 mM Hepes, 1 mM CaCl2, pH 7.4, and 20 µg/ml PMSF) and an equal volume of 600 mM NaCl. After centrifugation at 5,000 g for 3 min, the disrupted cells were separated into three layers, including the supernatant, proteinaceous gel, and precipitate.

Protocol tips

The recombinant T. thermophila strains were cultured at 5 × 105 cells/ml in Neff medium for 16 hr and treated with 15 µg/ml of cadmium chloride to induce transgene expression. T. thermophila cells were collected by centrifugation for 5 min at 1,000 g, and then resuspended in pre-chilled buffer A (40 mM Hepes, 1 mM CaCl2, pH 7.4, and 20 µg/ml PMSF) and an equal volume of 600 mM NaCl. After centrifugation at 5,000 g for 3 min, the disrupted cells were separated into three layers, including the supernatant, proteinaceous gel, and precipitate.

Publication protocol

"The recombinant T. thermophila strains were cultured at 5 × 105 cells/ml in Neff medium for 16 hr and treated with 15 µg/ml of cadmium chloride to induce transgene expression. T. thermophila cells were collected by centrifugation for 5 min at 1,000 g, and then resuspended in pre-chilled buffer A (40 mM Hepes, 1 mM CaCl2, pH 7.4, and 20 µg/ml PMSF) and an equal volume of 600 mM NaCl. After centrifugation at 5,000 g for 3 min, the disrupted cells were separated into three layers, including the supernatant, proteinaceous gel, and precipitate. The proteinaceous gel and precipitate were washed in 10 volumes of buffer A and centrifuged at 5,000 g for 3 min. Finally, the samples were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting.

Protein samples were loaded onto a 12% SDS-PAGE mini-gel under reducing conditions. The separated proteins were transferred onto a nitrocellulose membrane for 2 hr at 45 mA using a semi-dry transfer system (Bio-Rad) and blocked with Tris buffered saline (TBS, 150 mM NaCl, 20 mM Tris-HCl, pH 8.0) containing 3% bovine serum albumin overnight at 4°C. The recombinant NS1 protein was detected in transformed T. thermophila using specific rabbit antiserum (1:100). After washing with TBST (TBS with 0.05% Tween-20), the membrane was incubated with secondary goat anti-rabbit IgG antibodies conjugated to alkaline phosphatase (1:20,000) (Sigma-Aldrich, St. Louis, MO, U.S.A.) for 1 hr at 37°C. The membrane was then rinsed extensively and developed using BCIP/NBT substrate solution, until the protein bands became visible. The target protein bands on the gel were excised, and enzymatically digested into peptides by trypsin for liquid chromatography tandem mass spectrometry (LC-MS/MS). The data were searched using the MASCOT program (http://www.matrixscience.com). The procedure for purification of recombinant protein has been described previously [9]. The protein was purified by metal affinity chromatography and then dialyzed overnight. Finally, the protein was concentrated to 6 mg/ml using a 10 kDa Amicon Ultra-15 centrifugal filter tube (Merck Millipore, Ireland). Protein purity was assayed by SDS-PAGE."

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